Screening

ABSTRACT

A method of producing a conformational specific binding partner of a GPCR, the method comprising: a) providing a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR; b) providing a test compound; c) determining whether the test compound binds to the mutant GPCR when residing in a particular conformation; and d) isolating a test compound that binds to the mutant GPCR when residing in the particular conformation. Methods of producing GPCRs with increased stability relative to a parent GPCR are also disclosed.

The present invention relates to the screening of binding partners ofG-protein coupled receptors (GPCRs) and particularly to conformationspecific binding partners of GPCRs.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

GPCRs constitute a very large family of proteins that control manyphysiological processes and are the targets of many effective drugs.Thus, they are of considerable pharmacological importance. A list ofGPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, whichis incorporated herein by reference. GPCRs are generally unstable whenisolated, and despite considerable efforts, it has only been possible tocrystallise bovine rhodopsin, which naturally is exceptionally stableand the beta 2 adrenergic receptor which was crystallised as a fusionprotein or in complex with an antibody fragment.

GPCRs are druggable targets, and reference is made particularly toOverington et al (2006) Nature Rev. Drug Discovery 5, 993-996 whichindicates that over a quarter of present drugs have a GPCR as a target.

GPCRs are thought to exist in multiple distinct conformations which areassociated with different pharmacological classes of ligand such asagonists and antagonists, and to cycle between these conformations inorder to function (Kenakin T. (1997) Ann NY Acad Sci 812, 116-125).Switching between conformations contributes to the difficulty inobtaining crystal structures of receptors.

The generation of conformation specific binding partners to GPCRs ishindered by several factors. For example, GPCRs generally have poorstability when removed from their native membrane environment thatseverely restricts the range of conditions that can be explored withouttheir immediate denaturation or precipitation. The inability to producepurified GPCRs in their native conformation prevents their use in a widerange of screening paradigms which depend on the use of purifiedreceptors. Thus, GPCR screening has traditionally depended on assays inwhich the receptor is present in cell membranes or whole cells.

Many GPCRs represent important therapeutic targets which could beexploited by biotherapeutics such as antibodies. The generation oftherapeutic antibodies for GPCRs has been extremely difficult due inpart to the lack of a suitable immunogen. The usual route is to takesmall peptide fragments of the receptor for immunization however thesedo not retain their native conformation and often result in bindingpartners that can bind to and label the receptor but have no functionalagonist or antagonist activity. Due to the unique physical conformationof GPCRs it is also known that biotherapeutics such as antibodiesrecognise combinations of polypeptide ‘loops’, features that are lostwhen peptide fragments are used in isolation. It is well known that thelocal membrane environment of GPCRs maintains the tertiary conformationof the protein, and governs which epitopes are present on theextracellular surface. These epitopes can in theory be recognised bybiotherapeutics, however it is non-trivial to raise biotherapeutics,such as antibodies, to membranes or membrane fragments containing atarget GPCR as these preparations inevitably contain other non-targetGPCRs and membrane-associated proteins, and other membrane componentssuch as lipoproteins, apolipoproteins, lipids, phosophoinsositol lipidsand liposacharides which can act as non-desired antigens in thebiotherapeutic selection and production process.

We have developed a methodology for the stabilisation of a GPCR in aparticular biologically relevant conformation. Such receptors have anumber of advantages as immunogens and/or selection and screeningreagents for the generation of binding partners. In particular, they areuseful for the generation of conformation-specific binding partners,which will frequently have functional properties, and have previouslyproven very difficult to generate.

For example, the stability of native, correctly folded receptorthroughout the expression, solubilisation and purification stepsfacilitates a high yield of purified GPCR (milligram quantities fromlab-scale cell culture). Furthermore, the stability of folded protein ina range of detergents and solubilisation buffers and additives, withoutdistortion of function, enables optimisation of conditions forimmunization, for immobilization on solid surfaces without denaturation(e.g. plastic plates, resins, beads or slides, directly or via affinitytags such as poly-Histidine tags), for the purpose of antibodyproduction and screening or for library screening (such as affibody,antibody, phage or small molecule libraries). For library screening,reduction in non-specific binding by removal of membranous matter andcell-surface “sticky antigens” will give a major improvement insignal/noise. Use of short-chain detergents with highly polar or chargedhead groups (such as lauryldimethylamine-oxide, octyl D-glucoside oroctyl D-maltoside) is also made possible, which will reveal a greaterproportion of the antigenic surfaces of the GPCR which are masked bylonger-chain detergents such as n-dodecyl beta-D-maltoside (Bamber et alPNAS 103 (2006) 16224-16229). The trapping of particular functionalconformations of the receptor will also increase the likelihood ofgenerating conformation-specific, functional binding partners.

Accordingly, a first aspect of the invention provides a method ofproducing a binding partner of a GPCR, the method comprising:

-   -   a) providing a mutant GPCR of a parent GPCR, wherein the mutant        GPCR has increased stability in a particular conformation        relative to the parent GPCR;    -   b) providing one or more test compounds;    -   c) determining whether the or each test compound binds to the        mutant GPCR when residing in the particular conformation; and    -   d) isolating those test compounds that bind to the mutant GPCR        when residing in the particular conformation.

By “binding partner” we mean a molecule that binds to a particular GPCR.Preferably, the molecule binds selectively to the GPCR. For example, itis preferred if the binding partner has a K_(d) value (dissociationconstant) which is at least five or ten times lower (i.e. higheraffinity) than for at least one other GPCR, and preferably more than 100or 500 times lower. More preferably, the binding partner of a GPCR has aK_(d) value more than 1000 or 5000 times lower than for at least oneother GPCR. However, it will be appreciated that the limits will varydependent upon the nature of the binding partner. Thus, typically, forsmall molecule binding partners, the binding partner typically has aK_(d) value which is at least 50 times or 100 times lower than for atleast one other GPCR. Typically, for antibody binding partners, thebinding partner typically has a K_(d) value which is at least 500 or1000 times lower than for at least one other GPCR.

The use of stabilised GPCRs trapped in particular conformations willincrease the likelihood of producing conformation-specific bindingpartners. Accordingly, it is appreciated that the method may be used toproduce a conformation-specific binding partner of a GPCR. Thus, themethod may also be used to identify binding partners that havefunctional activity such as agonists or antagonists (or otherpharmacological categories) which will be determined by the conformationin which the GPCR was stabilised.

By “conformation specific”, we mean that the binding partner of the GPCRbinds selectively to a particular conformation of the GPCR and thus hasa K_(d) value for that conformation which is lower than for otherconformations of the same GPCR. Thus, a conformation specific bindingpartner will bind to one conformation of a GPCR (and may cause the GPCRto adopt this conformation), but does not bind as strongly to anotherconformation that the GPCR may be able to adopt. It will be appreciatedtherefore that, while the difference in affinity between twoconformations and the conformation specific binding partner may besmall, typically it will be sufficient to alter the equilibrium betweenconformational states and encourage the GPCR to adopt a particularconformation. Thus, a conformational specific binding partner may beconsidered to be one which traps a GPCR in a conformation of biologicalrelevance (e.g. ligand bound state). Typically, a conformation specificbinding partner has a K_(d) value (dissociation constant) which is atleast five or ten times lower (i.e. higher affinity) than for at leastone other conformation of the GPCR conformation, and preferably between100-10000 times lower.

Typically, the conformational specific binding partner binds to the GPCRwith a K_(d) of from mM to pM or from mM to fM, such as in the rangefrom μM to nM or in the range from nM to pM.

Kd values can be determined readily using methods well known in the artand as described, for example, below.

At equilibrium Kd=[R][L]/[RL]

where the terms in brackets represent the concentration of

-   -   Receptor-ligand complexes [RL],    -   unbound receptor [R], and    -   unbound (“free”) ligand [L].

In order to determine the Kd the value of these terms must be known.Since the concentration of receptor is not usually known then theHill-Langmuir equation is used where

Fractional occupancy=[L]/[L]+Kd.

In order to experimentally determine a Kd then, the concentration offree ligand and bound ligand at equilibrium must be known. Typically,this can be done by using a radio-labelled or fluorescently labelledligand which is incubated with the receptor (present in whole cells orhomogenised membranes) until equilibrium is reached. The amount of freeligand vs bound ligand must then be determined by separating the signalfrom bound vs free ligand. In the case of a radioligand this can be doneby centrifugation or filtration to separate bound ligand present onwhole cells or membranes from free ligand in solution. Alternatively ascintillation proximity assay is used. In this assay the receptor (inmembranes) is bound to a bead containing scintillant and a signal isonly detected by the proximity of the radioligand bound to the receptorimmobilised on the bead.

The affinity constant may also be determined in a functional assay(K_(B)). Here the receptor in a whole cell or membrane is activated byan agonist ligand and a response measured (e.g. mobilisation ofintracellular calcium, G protein activation, increase or decrease inadenylate cyclise or cAMP, activation of a signal transduction pathwaysuch as a MAP-kinase pathway or activation of gene transcription). Theability of an antagonist to inhibit agonist activity can be measured andfor a competitive antagonist is equal to the affinity constant.

The stability of the mutant GPCRs in a range of detergents, surfactantsand solubilisation buffers enables their purification outside of theirnormal membrane environment. Therefore, the GPCR can be provided in anisolated form removed from non-desired antigens such as non-targetGPCRs, membrane associated proteins and other membrane components suchas lipoproteins, apolipoproteins, lipis, phosphoinositol lipids andliposaccharides. Thus the method of the invention allows for theselection of a binding partner of a GPCR in the absence of suchnon-desired antigens. Thus the invention produces binding partners thathave enhanced selectivity for a GPCR over other membrane components.

Mutations of the parent GPCR that confer stability in a particularconformation are not expected to affect the binding of the parent GPCRresiding in a particular conformation to a particular binding partner.However, it is appreciated that once a binding partner has been isolatedby assessing binding to a mutant GPCR residing in a particularconformation, binding of that binding partner to the parent GPCRresiding in the same particular conformation may also be assessed.

Thus, in one embodiment the method further comprises:

-   -   (e) determining whether the or each test compound binds to the        parent GPCR when residing in the particular conformation; and    -   (f) isolating those test compounds that also bind the parent        GPCR when residing in the particular conformation.

Typically, the selected binding partner binds to the mutant GPCR whenresiding in a particular conformation with a similar potency to itsbinding to the parent GPCR when residing in the same particularconformation. Typically, the K_(d) values for the particular bindingpartner binding the mutant GPCR and the parent GPCR are within 5-10 foldof each other, such as within 2-3 fold. Typically, the binding of thebinding partner to the mutant GPCR compared to the parent GPCR would notbe more than 5 times weaker and not more than 10 times stronger.

Typically, the selected binding partner binds to the mutant GPCR withapproximately equal affinity (that is to say typically within 2-3 fold)or greater affinity than does the parent receptor, when residing in thesame conformation. For agonist-conformation mutants, the mutantstypically bind the agonist-conformation specific binding partners withthe same or higher affinity than the parent GPCR and typically bindantagonist-conformation specific binding partners with the same or loweraffinity than the parent GPCR. Similarly for antagonist-conformationmutants, the mutants typically bind the antagonist-conformation specificbinding partners with the same or higher affinity than the parent GPCRand typically bind agonist-conformation specific binding partners withthe same or lower affinity than the parent GPCR.

Selectivity of binding partners for particular conformations of GPCR orfor particular GPCRs and calculation of K_(d)s can be determined usingbinding assays well known in the art and as described, for example,below. Typically, K_(d) values are calculated using conventional GPCRassays in membranes wherein the binding affinity at different compoundconcentrations is measured. Examples of suitable assays include surfaceplasmon resonance assays and competition assays which are well known inthe art and are described below.

Typically, the mutant GPCR used in the present aspect is selected andprepared using any of the methods as described below.

Providing a Mutant GPCR of a Parent GPCR

A mutant GPCR with increased stability relative to its parent GPCR canbe provided by the methods described below, and by any of the methodsdisclosed in PCT applications WO 2008/114020 and PCT/GB2008/004032.

Method 1

A method for selecting a mutant G-protein coupled receptor (GPCR) withincreased stability, comprises

-   -   (a) providing one or more mutants of a parent GPCR,    -   (b) selecting a ligand, the ligand being one which binds to the        parent GPCR when the GPCR is residing in a particular        conformation,    -   (c) determining whether the or each mutant GPCR has increased        stability with respect to binding the selected ligand compared        to the stability of the parent GPCR with respect to binding that        ligand, and    -   (d) selecting those mutants that have an increased stability        compared to the parent GPCR with respect to binding of the        selected ligand.

The inventors have appreciated that, in order to improve the likelihoodof crystallisation of a GPCR in a biologically relevant form (which istherefore pharmacologically useful), it is desirable not only toincrease the stability of the protein, but also for the protein to havethis increased stability when in a particular conformation. Theconformation is determined by a selected ligand, and is a biologicallyrelevant conformation in particular a pharmacologically relevantconformation. Thus, the method of selection may be considered to be amethod for selecting mutants of a GPCR which have increased stability ofa particular conformation, for example they may have increasedconformational thermostability. The method may be used to create stable,conformationally locked GPCRs by mutagenesis. The selected mutant GPCRsare effectively purer forms of the parent molecules in that a muchhigher proportion of them occupies a particular conformational state.The deliberate selection of a chosen receptor conformation resolved fromother conformations by use of a ligand (or ligands) that bindpreferentially to this conformation is therefore an important feature ofthe selection method. The method may also be considered to be a methodfor selecting mutant GPCRs which are more tractable to crystallisation.This is because it is well known that decreased homogeneity or increasedpleiotropy within a population of molecules does not favourcrystallisation, and further, that an increased number of conformationsof a particular molecule does not favour crystallisation.

Thus a further method for selecting a mutant G-protein coupled receptor(GPCR) with increased stability, comprises

-   -   (a) providing one or more mutants of a parent GPCR,    -   (b) selecting a ligand, the ligand being one which binds to the        parent GPCR when the GPCR is residing in a particular        conformation,    -   (c) determining whether the or each mutant GPCR when residing in        the particular conformation has increased stability with respect        to binding the selected ligand compared to the stability of the        parent GPCR when residing in the same particular conformation        with respect to binding that ligand, and    -   (d) selecting those mutants that have an increased stability        compared to the parent GPCR with respect to binding of the        selected ligand.

In a review of the druggable genome by Hopkins & Groom (2002) NatureRev. Drug Discovery 1, 727-730, Table 1 contains a list of proteinfamilies many of which are GPCRs. Overington et al (2006) Nature Rev.Drug Discovery 5, 993-996 provides more details of drug targets, andFIG. 1 indicates that more than a quarter of current drugs target GPCRs.There are 52 GPCR targets for orally available drugs out of a total of186 total targets in this category.

Suitable GPCRs for use in the practice of the invention include, but arenot limited to β-adrenergic receptor, adenosine receptor, in particularadenosine A_(2a) receptor, and neurotensin receptor (NTR). Othersuitable GPCRs are well known in the art and include those listed inHopkins & Groom supra. In addition, the International Union ofPharmacology produce a list of GPCRs (Foord et al (2005) Pharmacol. Rev.57, 279-288, incorporated herein by reference and this list isperiodically updated athttp://www.iuphar-db.org/GPCR/ReceptorFamiliesForward). It will be notedthat GPCRs are divided into different classes, principally based ontheir amino acid sequence similarities. They are also divided intofamilies by reference to the natural ligands to which they bind. AllGPCRs are included in the scope of the invention.

The amino acid sequences (and the nucleotide sequences of the cDNAswhich encode them) of many GPCRs are readily available, for example byreference to GenBank. In particular, Foord et al supra gives the humangene symbols and human, mouse and rat gene IDs from Entrez Gene(http://www.ncbi.nlm.nih.gov/entrez). It should be noted, also, thatbecause the sequence of the human genome is substantially complete, theamino acid sequences of human GPCRs can be deduced therefrom.

Although the GPCR may be derived from any source, it is particularlypreferred if it is from a eukaryotic source. It is particularlypreferred if it is derived from a vertebrate source such as a mammal ora bird. It is particularly preferred if the GPCR is derived from rat,mouse, rabbit or dog or non-human primate or man, or from chicken orturkey. For the avoidance of doubt, we include within the meaning of“derived from” that a cDNA or gene was originally obtained using geneticmaterial from the source, but that the protein may be expressed in anyhost cell subsequently. Thus, it will be plain that a eukaryotic GPCR(such as an avian or mammalian GPCR) may be expressed in a prokaryotichost cell, such as E. coli, but be considered to be avian- ormammalian-derived, as the case may be.

In some instances, the GPCR may be composed of more than one differentsubunit. For example, the calcitonin gene-related peptide receptorrequires the binding of a single transmembrane helix protein (RAMP1) toacquire its physiological ligand binding characteristics. Effector,accessory, auxiliary or GPCR-interacting proteins which combine with theGPCR to form or modulate a functional complex are well known in the artand include, for example, receptor kinases, G-proteins and arrestins(Bockaert et at (2004) Curr Opinion Drug Discov and Dev 7, 649-657).

The mutants of the parent GPCR may be produced in any suitable way andprovided in any suitable form. Thus, for example, a series of specificmutants of the parent protein may be made in which each amino acidresidue in all or a part of the parent protein is independently changedto another amino acid residue. For example, it may be convenient to makemutations in those parts of the protein which are predicted to bemembrane spanning. The three-dimensional structure of rhodopsin is known(Li at al (2004) J Mol Biol 343, 1409-1438; Palczewski at al (2000)Science 289, 739-745), and it is possible to model certain GPCRs usingthis structure. Thus, conveniently, parts of the GPCR to mutate may bebased on modelling. Similarly, computer programs are available whichmodel transmembrane regions of GPCRs based on hydrophobicity (Kyle &Dolittle (1982) J. Mol. Biol. 157, 105-132), and use can be made of suchmodels when selecting parts of the protein to mutate. Conventionalsite-directed mutagenesis may be employed, or polymerase chainreaction-based procedures well known in the art may be used. It ispossible, but less desirable, to use ribosome display methods in theselection of the mutant protein.

Typically, each selected amino acid is replaced by Ala (ie Ala-scanningmutagenesis), although it may be replaced by any other amino acid. Ifthe selected amino acid is Ala, it may conveniently be replaced by Leu.Alternatively, the amino acid may be replaced by Gly (ie Gly-scanningmutagenesis), which may allow a closer packing of neighbouring helicesthat may lock the protein in a particular conformation. If the selectedamino acid is Gly, it may conveniently be replaced by Ala.

Although the amino acid used to replace the given amino acid at aparticular position is typically a naturally occurring amino acid,typically an “encodeable” amino acid, it may be a non-natural amino acid(in which case the protein is typically made by chemical synthesis or byuse of non-natural amino-acyl tRNAs). An “encodeable” amino acid is onewhich is incorporated into a polypeptide by translation of mRNA. It isalso possible to create non-natural amino acids or introduce non-peptidelinkages at a given position by covalent chemical modification, forexample by post-translational treatment of the protein or semisynthesis.These post-translational modifications may be natural, such asphosphorylation, glycosylation or palmitoylation, or synthetic orbiosynthetic.

Alternatively, the mutants may be produced by a random mutagenesisprocedure, which may be of the whole protein or of a selected portionthereof. Random mutagenesis procedures are well known in the art.

Conveniently, the mutant GPCR has one replaced amino acid compared tothe parent protein (ie it is mutated at one amino acid position). Inthis way, the contribution to stability of a single amino acidreplacement may be assessed. However, the mutant GPCR assayed forstability may have more than one replaced amino acid compared to theparent protein, such as 2 or 3 or 4 or 5 or 6 replacements.

As is discussed in more detail below, combinations of mutations may bemade based on the results of the selection method. It has been foundthat in some specific cases combining mutations in a single mutantprotein leads to a further increase in stability. Thus, it will beappreciated that the method of selection can be used in an iterative wayby, for example, carrying it out to identify single mutations whichincrease stability, combining those mutations in a single mutant GPCRswhich is the GPCR then provided in part (a) of the method. Thus,multiply-mutated mutant proteins can be selected using the method.

The parent GPCR need not be the naturally occurring protein.Conveniently, it may be an engineered version which is capable ofexpression in a suitable host organism, such as Escherichia coli. Forexample, as described in Example 1, a convenient engineered version ofthe turkey β-adrenergic receptor is one which is truncated and lacksresidues 1-33 of the amino acid sequence (ie βAR₃₄₋₄₂₄). The parent GPCRmay be a truncated form of the naturally occurring protein (truncated ateither or both ends), or it may be a fusion, either to the naturallyoccurring protein or to a fragment thereof. Alternatively oradditionally, the parent GPCR, compared to a naturally-occurring GPCR,may be modified in order to improve, for example, solubility,proteolytic stability (eg by truncation, deletion of loops, mutation ofglycosylation sites or mutation of reactive amino acid side chains suchas cysteine). In any event, the parent GPCR is a protein that is able tobind to the selected ligand which ligand is one which is known to bindthe naturally occurring GPCR. Conveniently, the parent GPCR is onewhich, on addition of an appropriate ligand, can affect any one or moreof the downstream activities which are commonly known to be affected byG-protein activation.

However, it will be appreciated that the stability of the mutant is tobe compared to a parent in order to be able to assess an increase instability.

A ligand is selected, the ligand being one which binds to the parentGPCR when residing in a particular conformation. Typically, the ligandwill bind to one conformation of the parent GPCR (and may cause the GPCRto adopt this conformation), but does not bind as strongly to anotherconformation that the GPCR may be able to adopt. Thus, the presence ofthe ligand may be considered to encourage the GPCR to adopt theparticular conformation. Thus, the method may be considered to be a wayof selecting mutant GPCRs which are trapped in a conformation ofbiological relevance (eg ligand bound state), and which are more stablewith respect to that conformation.

Preferably the particular conformation in which the GPCR resides in step(c) corresponds to the class of ligand selected in step (b).

Preferably the selected ligand is from the agonist class of ligands andthe particular conformation is an agonist conformation, or the selectedligand is from the antagonist class of ligands and the particularconformation is an antagonist conformation.

Preferably the selected ligand is from the agonist class of ligands andthe particular conformation in which the GPCR resides in step (c) is theagonist conformation.

Preferably, the selected ligand binding affinity for the mutant receptorshould be equal to or greater than that for the wild type receptor;mutants that exhibit significantly reduced binding to the selectedligand are typically rejected.

By “ligand” we include any molecule which binds to the GPCR and whichcauses the GPCR to reside in a particular conformation. The ligandpreferably is one which causes more than half of the GPCR moleculesoverall to be in a particular conformation.

Many suitable ligands are known.

Typically, the ligand is a full agonist and is able to bind to the GPCRand is capable of eliciting a full (100%) biological response, measuredfor example by G-protein coupling, downstream signalling events or aphysiological output such as vasodilation. Thus, typically, thebiological response is GDP/GTP exchange in a G-protein, followed bystimulation of the linked effector pathway. The measurement, typically,is GDP/GTP exchange or a change in the level of the end product of thepathway (eg cAMP, cGMP or inositol phosphates). The ligand may also be apartial agonist and is able to bind to the GPCR and is capable ofeliciting a partial (<100%) biological response.

The ligand may also be an inverse agonist, which is a molecule whichbinds to a receptor and reduces its basal (ie unstimulated by agonist)activity sometimes even to zero.

The ligand may also be an antagonist, which is a molecule which binds toa receptor and blocks binding of an agonist, so preventing a biologicalresponse. Inverse agonists and partial agonists may under certain assayconditions be antagonists.

The above ligands may be orthosteric, by which we include the meaningthat they combine with the same site as the endogenous agonist; or theymay be allosteric or allotopic, by which we include the meaning thatthey combine with a site distinct from the orthosteric site. The aboveligands may be syntopic, by which we include the meaning that theyinteract with other ligand(s) at the same or an overlapping site. Theymay be reversible or irreversible.

In relation to antagonists, they may be surmountable, by which weinclude the meaning that the maximum effect of agonist is not reduced byeither pre-treatment or simultaneous treatment with antagonist; or theymay be insurmountable, by which we include the meaning that the maximumeffect of agonist is reduced by either pre-treatment or simultaneoustreatment with antagonist; or they may be neutral, by which we includethe meaning the antagonist is one without inverse agonist or partialagonist activity. Antagonists typically are also inverse agonists.

Ligands for use in the selection method may also be allostericmodulators such as positive allosteric modulators, potentiators,negative allosteric modulators and inhibitors. They may have activity asagonists or inverse agonists in their own right or they may only haveactivity in the presence of an agonist or inverse agonist in which casethey are used in combination with such molecules in order to bind to theGPCR.

Neubig et al (2003) Pharmacol. Rev. 55, 597-606, incorporated herein byreference, describes various classes of ligands.

Preferably, the above-mentioned ligands are small organic or inorganicmoieties, but they may be peptides or polypeptides. Typically, when theligand is a small organic or organic moiety, it has a M_(r) of from 50to 2000, such as from 100 to 1000, for example from 100 to 500.

Typically, the ligand binds to the GPCR with a K_(d) of from mM to pM,such as in the range of from μM (micromolar) to nM. Generally, theligands with the lowest Kd are preferred.

Small organic molecule ligands are well known in the art, for examplesee the Examples below. Other small molecule ligands include 5HT whichis a full agonist at the 5HT1A receptor; eltoprazine which is a partialagonist at the 5HT1A receptor (see Newman-Tancredi et al (1997)Neurophamacology 36, 451-459); (+)-butaclamol and spiperone are dopamineD2 receptor inverse agonists (see Roberts & Strange (2005) Br. J.Pharmacol. 145, 34-42); and WIN55212-3 is a neutral antagonist of CB2(Savinainen et al (2005) Br. J. Pharmacol. 145, 636-645).

The ligand may be a peptidomimetic, a nucleic acid, a peptide nucleicacid (PNA) or an aptamer. It may be an ion such as Na⁺ or Zn²⁺, a lipidsuch as oleamide, or a carbohydrate such as heparin.

The ligand may be a polypeptide which binds to the GPCR. Suchpolypeptides (by which we include oligopeptides) are typically fromM_(r) 500 to M_(r) 50,000, but may be larger. The polypeptide may be anaturally occurring GPCR-interacting protein or other protein whichinteracts with the GPCR, or a derivative or fragment thereof, providedthat it binds selectively to the GPCR in a particular conformation.GPCR-interacting proteins include those associated with signalling andthose associated with trafficking, which often act via PDZ domains inthe C terminal portion of the GPCR.

Polypeptides which are known to bind certain GPCRs include any of a Gprotein, an arrestin, a RGS protein, G protein receptor kinase, a RAMP,a 14-3-3 protein, a NSF, a periplakin, a spinophilin, a GPCR kinase, areceptor tyrosine kinase, an ion channel or subunit thereof, an ankyrinand a Shanks or Horner protein. Other polypeptides include NMDA receptorsubunits NR1 or NR2a, calcyon, or a fibronectin domain framework. Thepolypeptide may be one which binds to an extracellular domain of a GPCR,such as fibulin-1. The polypeptide may be another GPCR, which binds tothe selected GPCR in a hetero-oligomer. A review of protein-proteininteractions at GPCRs is found in Milligan & White (2001) TrendsPharmacol. Sci. 22, 513-518, or in Bockaert et al (2004) Curr. OpinionDrug Discov. Dev. 7, 649-657 incorporated herein by reference.

The polypeptide ligand may conveniently be an antibody which binds tothe GPCR. By the term “antibody” we include an immunoglobulin whethernatural or partly or wholley synthetically produced. Examples includeimmunoglobulin isotypes and their isotypic subclasses, and monoclonalantibodies and fragments thereof comprising an antigen binding domainssuch as Fab, F(ab′)₂, single chain Fv (scFv), Fv, domain antibodies(dAbs) and diabodies. Mention is also made of camelid antibodies andengineered camelid antibodies. Such molecules which bind GPCRs are knownin the art and in any event can be made using well known technology.Suitable antibodies include ones presently used in radioimmunoassay(RIAs) for GPCRs since they tend to recognise conformational epitopes.

The polypeptide may also be a binding protein based on a modularframework, such as ankyrin repeat proteins, armadillo repeat proteins,leucine rich proteins, tetratriopeptide repeat proteins or DesignedAnkyrin Repeat Proteins (DARPins) or proteins based on lipocalin orfibronectin domains or Affilin scaffolds based on either human gammacrystalline or human ubiquitin.

In one embodiment of the selection method, the ligand is covalentlyjoined to the GPCR, such as a G-protein or arrestin fusion protein. SomeGPCRs (for example thrombin receptor) are cleaved N-terminally by aprotease and the new N-terminus binds to the agonist site. Thus, suchGPCRs are natural GPCR-ligand fusions.

It will be appreciated that the use of antibodies, or other “universal”binding polypeptides (such as G-proteins which are known to couple withmany different GPCRs) may be particularly advantageous in the use of themethod on “orphan” GPCRs for which the natural ligand, and smallmolecule ligands, are not known.

Once the ligand has been selected, it is then determined whether the oreach mutant GPCR has increased stability with respect to binding theselected ligand compared to the parent GPCR with respect to binding thatligand. It will be appreciated that this step (c) is one in which it isdetermined whether the or each mutant GPCR has an increased stability(compared to its parent) for the particular conformation which isdetermined by the selected ligand. Thus, the mutant GPCR has increasedstability with respect to binding the selected ligand as measured byligand binding or whilst binding the selected ligand. As is discussedbelow, it is particularly preferred if the increased stability isassessed whilst binding the selected ligand.

The increased stability is conveniently measured by an extended lifetimeof the mutant under the imposed conditions which may lead to instability(such as heat, harsh detergent conditions, chaotropic agents and so on).Destabilisation under the imposed condition is typically determined bymeasuring denaturation or loss of structure. As is discussed below, thismay manifest itself by loss of ligand binding ability or loss ofsecondary or tertiary structure indicators.

As is described with respect to FIG. 12 below (which depicts aparticular, preferred embodiment), there are different assay formatswhich may be used to determine stability of the mutant GPCR.

In one embodiment the mutant GPCR may be brought into contact with aligand before being subjected to a procedure in which the stability ofthe mutant is determined (the mutant GPCR and ligand remaining incontact during the test period). Thus, for example, when the method isbeing used to select for mutant GPCRs which in one conformation bind toa ligand and which have improved thermostablity, the receptor iscontacted with the ligand before being heated, and then the amount ofligand bound to the receptor following heating may be used to expressthermostability compared to the parent receptor. This provides a measureof the amount of the GPCR which retains ligand binding capacityfollowing exposure to the denaturing conditions (eg heat), which in turnis an indicator of stability.

In an alternative (but less preferred) embodiment, the mutant GPCR issubjected to a procedure in which the stability of the mutant isdetermined before being contacted with the ligand. Thus, for example,when the method is being used to select for mutant membrane receptorswhich in one conformation bind to a ligand and which have improvedthermostability, the receptor is heated first, before being contactedwith the ligand, and then the amount of ligand bound to the receptor maybe used to express thermostability. Again, this provides a measure ofthe amount of the GPCR which retains ligand binding capacity followingexposure to the denaturing conditions.

In both embodiments, it will be appreciated that the comparison ofstability of the mutant is made by reference to the parent moleculeunder the same conditions.

It will be appreciated that in both of these embodiments, the mutantsthat are selected are ones which have increased stability when residingin the particular conformation compared to the parent protein.

The preferred route may be dependent upon the specific GPCR, and will bedependent upon the number of conformations accessible to the protein inthe absence of ligand. In the embodiment described in FIG. 12, it ispreferred if the ligand is present during the heating step because thisincreases the probability that the desired conformation is selected.

From the above, it will be appreciated that the selection methodincludes a method for selecting a mutant GPCR with increasedthermostability, the method comprising (a) providing one or more mutantsof a parent GPCR, (b) selecting an antagonist or an agonist which bindsthe parent GPCR, (c) determining whether the or each mutant hasincreased thermostability when in the presence of the said antagonist oragonist by measuring the ability of the mutant GPCR to bind the selectedsaid antagonist or agonist at a particular temperature and after aparticular time compared to the parent GPCR and (d) selecting thosemutant GPCRs that bind more of the selected said antagonist or agonistat the particular temperature and after the particular time than theparent GPCR under the same conditions. In step (c), a fixed period oftime at the particular temperature is typically used in measuring theability of the mutant GPCR to bind the selected said antagonist oragonist. In step (c), typically a temperature and a time is chosen atwhich binding of the selected said antagonist or agonist by the parentGPCR is reduced by 50% during the fixed period of time at thattemperature (which is indicative that 50% of the receptor isinactivated; “quasi” Tm).

Conveniently, when the ligand is used to assay the GPCR (ie used todetermine if it is in a non-denatured state), the ligand is detectablylabelled, eg radiolabelled or fluorescently labelled. In anotherembodiment, ligand binding can be assessed by measuring the amount ofunbound ligand using a secondary detection system, for example anantibody or other high affinity binding partner covalently linked to adetectable moiety, for example an enzyme which may be used in acolorimetric assay (such as alkaline phosphatase or horseradishperoxidase). FRET methodology may also be used. It will be appreciatedthat the ligand used to assay the mutant GPCR in determining itsstability need not be the same ligand as selected in step (b) of themethod.

Although it is convenient to measure the stability of the parent andmutant GPCR by using the ability to bind a ligand as an indicator of thepresence of a non-denatured protein, other methods are known in the art.For example, changes in fluorescence spectra can be a sensitiveindicator of unfolding, either by use of intrinsic tryptophanfluorescence or the use of extrinsic fluorescent probes such as1-anilino-8-napthaleneulfonate (ANS), for example as implemented in theThermofluor™ method (Mezzasalma et al, J Biomol Screening, 2007, April;12(3):418-428). Proteolytic stability, deuterium/hydrogen exchangemeasured by mass spectrometry, blue native gels, capillary zoneelectrophoresis, circular dichroism (CD) spectra and light scatteringmay also be used to measure unfolding by loss of signals associated withsecondary or tertiary structure. However, all these methods require theprotein to be purified in reasonable quantities before they can be used(eg high pmol/nmol quantities), whereas the method described in theExamples makes use of pmol amounts of essentially unpurified GPCR.

In a preferred embodiment, in step (b) two or more ligands of the sameclass are selected, the presence of each causing the GPCR to reside inthe same particular conformation. Thus, in this embodiment, one or moreligands (whether natural or non-natural) of the same class (eg fullagonist or partial agonist or antagonist or inverse agonist) may beused. Including multiple ligands of the same class in this process,whether in series or in parallel, minimises the theoretical risk ofinadvertently engineering and selecting multiply mutated receptorconformations substantially different to the parent, for example intheir binding site, but still able, due to compensatory changes, to bindligand. The following steps may be used to mitigate this risk:

1. Select a chemically distinct set (eg n=2-5) of ligands, in a commonpharmacological class as evidenced by for example a binding orfunctional or spectroscopic assay. These ligands should be thought tobind to a common spatial region of the receptor, as evidenced forexample by competitive binding studies using wild type and/or mutatedreceptors, and/or by molecular modelling, although they will notnecessarily express a common pharmacophore.2. Make single or multiple receptor mutants intended to increasestability, and assay for tight binding using the full set of ligands.The assays can be parallelised, multiplexed or run in series.3. Confirm authenticity of stabilised receptor mutant by measurement forexample of the binding isotherm for each ligand, and by measurement ofthe stability shift with ligand (the window should typically be narrowedcompared to wild type).

In order to guard against changes in apparent affinity caused byperturbations to the binding site upon mutation, preferably ligands ofthe same pharmacological class, but different chemical class, should beused to profile the receptor. These should typically show similar shiftsin affinity (mutant versus parent, e.g. wild type) in spite of havingdifferent molecular recognition properties. Binding experiments shouldpreferably be done using labelled ligand within the same pharmacologicalclass.

Nonetheless it should be recognised that conformational substrates mayexist that are specific to chemical classes of ligand within the samepharmacological class, and these may be specifically stabilised in theprocedure depending on the chemical class of the selected ligand.

Typically the selected ligand binds to the mutant GPCR with a similarpotency to its binding to the parent GPCR. Typically, the K_(d) valuesfor the particular ligand binding the mutant GPCR and the parent GPCRare within 5-10 fold of each other, such as within 2-3 fold. Typically,the binding of the ligand to the mutant GPCR compared to the parent GPCRwould be not more than 5 times weaker and not more than 10 timesstronger.

Typically, mutant receptors which have been stabilised in the selectedconformation should bind the selected ligand with approximately equalaffinity (that is to say typically within 2-3 fold) or greater affinitythan does the parent receptor. For agonist-conformation mutants, themutants typically bind the agonists with the same or higher affinitythan the parent GPCR and typically bind antagonists with the same orlower affinity than the parent GPCR. Similarly forantagonist-conformation mutants, the mutants typically bind theantagonists with the same or higher affinity than the parent GPCR andtypically bind agonists with the same or lower affinity than the parentGPCR.

Mutants that exhibit a significant reduction (typically greater than 2-3fold) in affinity for the selecting ligand are typically rejected.

Typically, the rank order of binding of a set of ligands of the sameclass are comparable, although there may be one or two reversals in theorder, or there may be an out-tier from the set.

In a further embodiment, two or more ligands that bind simultaneously tothe receptor in the same conformation may be used, for example anallosteric modulator and orthosteric agonist.

For the avoidance of doubt, and as is evident from the Examples, it isnot necessary to use multiple ligands for the method to be effective.

In a further embodiment, it may be advantageous to select those mutantGPCRs which, while still being able to bind the selected ligand, are notable to bind, or bind less strongly than the parent GPCR, a secondselected ligand which is in a different class to the first ligand. Thus,for example, the mutant GPCR may be one that is selected on the basisthat it has increased stability with respect to binding a selectedantagonist, but the mutant GPCR so selected is further tested todetermine whether it binds to a full agonist (or binds less strongly toa full agonist than its parent GPCR). Mutants are selected which do notbind (or have reduced binding of) the full agonist. In this way, furtherselection is made of a GPCR which is locked into one particularconformation.

It will be appreciated that the selected ligand (with respect to part(b) of the method) and the further (second) ligand as discussed above,may be any pair of ligand classes, for example: antagonist and fullagonist; full agonist and antagonist; antagonist and inverse agonist;inverse agonist and antagonist; inverse agonist and full agonist; fullagonist and inverse agonist; and so on.

It is preferred that the mutant receptor binds the further (second)ligand with an affinity which is less than 50% of the affinity theparent receptor has for the same further (second) ligand, morepreferably less than 10% and still more preferably less than 1% or 0.1%or 0.01% of affinity for the parent receptor. Thus, the K_(d) for theinteraction of the second ligand with mutant receptor is higher than forthe parent receptor. As is shown in Example 1, the mutant β-adrenergicreceptor βAR-m23 (which was selected by the selection method using anantagonist) binds an agonist 3 orders of magnitude more weakly than itsparent (ie K_(d) is 1000×higher). Similarly, in Example 2, the mutantadenosine A2a receptor Rant21 binds agonist 2-4 orders of magnitude moreweakly than its parent.

This type of counter selection is useful because it can be used todirect the mutagenesis procedure more specifically (and therefore morerapidly and more efficiently) along a pathway towards a pureconformation as defined by the ligand.

Preferably, the mutant GPCR is provided in a suitable solubilised formin which it maintains structural integrity and is in a functional form(eg is able to bind ligand). An appropriate solubilising system, such asa suitable detergent (or other amphipathic agent) and buffer system isused, which may be chosen by the person skilled in the art to beeffective for the particular protein. Typical detergents which may beused include, for example, dodecylmaltoside (DDM) or CHAPS oroctylglucoside (OG) or many others. It may be convenient to includeother compounds such as cholesterol hemisuccinate or cholesterol itselfor heptane-1,2,3-triol. The presence of glycerol or proline or betainemay be useful. It is important that the GPCR, once solubilised from themembrane in which it resides, must be sufficiently stable to be assayed.For some GPCRs, DDM will be sufficient, but glycerol or other polyolsmay be added to increase stability for assay purposes, if desired.Further stability for assay purposes may be achieved, for example, bysolubilising in a mixture of DDM, CHAPS and cholesterol hemisuccinate,optionally in the presence of glycerol. For particularly unstable GPCRs,it may be desirable to solubilise them using digitonin or amphipols orother polymers which can solubilise GPCRs directly from the membrane, inthe absence of traditional detergents and maintain stability typicallyby allowing a significant number of lipids to remain associated with theGPCR. Nanodiscs may also be used for solubilising extremely unstablemembrane proteins in a functional form.

Typically, the mutant GPCR is provided in a crude extract (eg of themembrane fraction from the host cell in which it has been expressed,such as E. coli). It may be provided in a form in which the mutantprotein typically comprises at least 75%, more typically at least 80% or85% or 90% or 95% or 98% or 99% of the protein present in the sample. Ofcourse, it is typically solubilised as discussed above, and so themutant GPCR is usually associated with detergent molecules and/or lipidmolecules.

A mutant GPCR may be selected which has increased stability to anydenaturant or denaturing condition such as to any one or more of heat, adetergent, a chaotropic agent or an extreme of pH.

In relation to an increased stability to heat (ie thermostability), thiscan readily be determined by measuring ligand binding or by usingspectroscopic methods such as fluorescence, CD or light scattering at aparticular temperature. Typically, when the GPCR binds to a ligand, theability of the GPCR to bind that ligand at a particular temperature maybe used to determine thermostability of the mutant. It may be convenientto determine a “quasi T_(m)” ie the temperature at which 50% of thereceptor is inactivated under stated conditions after incubation for agiven period of time (eg 30 minutes). Mutant GPCRs of higherthermostability have an increased quasi Tm compared to their parents.

In relation to an increased stability to a detergent or to a chaotrope,typically the GPCR is incubated for a defined time in the presence of atest detergent or a test chaotropic agent and the stability isdetermined using, for example, ligand binding or a spectroscopic methodas discussed above.

In relation to an extreme of pH, a typical test pH would be chosen (egin the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

Because relatively harsh detergents are used during crystallisationprocedures, it is preferred that the mutant GPCR is stable in thepresence of such detergents. The order of “harshness” of certaindetergents is DDM, C₁₁→C₁₀→C₉→C₈ maltoside or glucoside,lauryldimethylamine oxide (LDAO) and SDS. It is particularly preferredif the mutant GPCR is more stable to any of C₉ maltoside or glucoside,C₈ maltoside or glucoside, LDAO and SDS, and so it is preferred thatthese detergents are used for stability testing.

Because of its ease of determination, it is preferred thatthermostability is determined, and those mutants which have an increasedthermostability compared to the parent protein with respect to theselected condition are chosen. It will be appreciated that heat isacting as the denaturant, and this can readily be removed by cooling thesample, for example by placing on ice. It is believed thatthermostability may also be a guide to the stability to otherdenaturants or denaturing conditions. Thus, increased thermostability islikely to translate into stability in denaturing detergents, especiallythose that are more denaturing than DDM, eg those detergents with asmaller head group and a shorter alkyl chain and/or with a charged headgroup. We have found that a thermostable GPCR is also more stabletowards harsh detergents.

When an extreme of pH is used as the denaturing condition, it will beappreciated that this can be removed quickly by adding a neutralisingagent. Similarly, when a chaotrope is used as a denaturant, thedenaturing effect can be removed by diluting the sample below theconcentration in which the chaotrope exerts its chaotropic effect.

In a particular embodiment of the selection method, the GPCR isβ-adrenergic receptor (for example from turkey) and the ligand isdihydroalprenolol (DHA), an antagonist.

In a further preferred embodiment of the selection method, the GPCR isthe adenosine A_(2a) receptor (A_(2a)R) (for example, from man) and theligand is ZM 241385 (4-[2-[[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-α][1,3,5]triazin-5-yl]amino]ethyl]phenol), anantagonist or NECA (5′-N-ethylcarboxamido adenosine), an agonist.

In a still further preferred embodiment of the selection method, theGPCR is the neurotensin receptor (NTR) (for example, from rat) and theligand is neurotensin, an agonist.

Method 2

A method for preparing a mutant GPCR selected as above comprises:

-   -   (a) carrying out a method to select a mutant GPCR as described        above,    -   (b) identifying the position or positions of the mutated amino        acid residue or residues in the mutant GPCR or GPCRs which has        been selected for increased stability, and    -   (c) synthesising a mutant GPCR which contains a mutation at one        or more of the positions identified.

As can be seen in the Examples, surprisingly, changes to a single aminoacid within the GPCR may increase the stability of the protein comparedto the parent protein with respect to a particular condition in whichthe protein resides in a particular conformation. Thus, in oneembodiment of the method of the second aspect of the invention, a singleamino acid residue of the parent protein is changed in the mutantprotein. Typically, the amino acid residue is changed to the amino acidresidue found in the mutant tested in the method of the first aspect ofthe invention. However, it may be replaced by any other amino acidresidue, such as any naturally-occurring amino acid residue (inparticular, a “codeable” amino acid residue) or a non-natural aminoacid. Generally, for convenience, the amino acid residue is replacedwith one of the 19 other codeable amino acids. Preferably, it is thereplaced amino acid residue which is present in the mutant selected inthe selection method described above.

Also as can be seen in the Examples, a further increase in stability maybe obtained by replacing more than one of the amino acids of the parentprotein. Typically, each of the amino acids replaced is one which hasbeen identified using the selection method described above. Typically,each amino acid identified is replaced by the amino acid present in themutant protein although, as noted above, it may be replaced with anyother amino acid.

Typically, the mutant GPCR contains, compared to the parent protein,from 1 to 10 replaced amino acids, preferably from 1 to 8, typicallyfrom 2 to 6 such as 2, 3, 4, 5 or 6 replaced amino acids.

It will be appreciated that the multiple mutants may be subject to theselection method. In other words, multiple mutants may be provided instep (a) of the selection. It will be appreciated that multiplymutagenised GPCRs may be made, whose conformation has been selected tocreate a very stable multiple point mutant protein.

The mutant GPCRs may be prepared by any suitable method. Conveniently,the mutant protein is encoded by a suitable nucleic acid molecule andexpressed in a suitable host cell. Suitable nucleic acid moleculesencoding the mutant GPCR may be made using standard cloning techniques,site-directed mutagenesis and PCR as is well known in the art. Suitableexpression systems include constitutive or inducible expression systemsin bacteria or yeasts, virus expression systems such as baculovirus,semliki forest virus and lentiviruses, or transient transfection ininsect or mammalian cells. Suitable host cells include E. coli,Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Spodoptera frugiperda and Trichoplusiani cells.Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 andso on. It is known that some GPCRs require specific lipids (egcholesterol) to function. In that case, it is desirable to select a hostcell which contains the lipid. Additionally or alternatively the lipidmay be added during isolation and purification of the mutant protein. Itwill be appreciated that these expression systems and host cells mayalso be used in the provision of the mutant GPCR in part (a) of theselection method.

Molecular biological methods for cloning and engineering genes andcDNAs, for mutating DNA, and for expressing polypeptides frompolynucleotides in host cells are well known in the art, as exemplifiedin “Molecular cloning, a laboratory manual”, third edition, Sambrook, J.& Russell, D. W. (eds), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference.

It is appreciated that it may be determined whether the selected orprepared mutant GPCR is able to couple to a G protein. It is alsopreferred if it is determined whether the selected or prepared mutantGPCR is able to bind a plurality of ligands of the same class as theselecting ligand with a comparable spread and/or rank order of affinityas the parent GPCR.

Method 3

As shown in Examples 1-3 and described above, thermostabilisingmutations are scattered widely throughout the sequences of the turkeybeta1 adrenergic receptor, human adenosine receptor, rat neurotensinreceptor and human muscarinic receptor. FIG. 17 provides an alignment ofthese sequences with the sequence of the human beta-2AR such that whenthe thermostabilising mutations are positioned onto the sequences then,in 11 instances out of a total of 70, two sequences contain mutations atthe same position (denoted in FIG. 17 with a star). Thus it will beappreciated that once one or more stabilising mutations have beenidentified in one GPCR, a further GPCR with increased stability can begenerated by aligning the amino acid sequences of the GPCRs and makingthe same one or more mutations at the corresponding position orpositions. This concept is clearly exemplified in FIG. 26 wherein thesix thermostabilising mutations in turkey β1-m23 were transferreddirectly to the human β2 receptor. The resultant mutant, β2-m23, had aTm 12° C. higher than that of the human β2 receptor.

Accordingly, a further method for producing a mutant GPCR with increasedstability relative to its parent GPCR comprises

-   -   (i) identifying in the amino acid sequence of one or more        mutants of a first parent GPCR with increased stability relative        to the first parent GPCR, the position or positions at which the        one or more mutants have at least one different amino acid        residue compared to the first parent GPCR, and    -   (ii) making one or more mutations in the amino acid sequence        that defines a second GPCR at the corresponding position or        positions, to provide one or more mutants of a second parent        GPCR with increased stability relative to the second parent        GPCR.

The one or more mutants of a first parent GPCR may be selected orprepared according to the selection or preparation methods describedabove. Hence, this method may also be used to create stable,conformationally locked GPCRs by mutagenesis. For example, following theselection of mutant GPCRs which have increased stability in a particularconformation, the stabilising mutation can be identified and the aminoacid at a corresponding position in a second GPCR replaced to produce amutant GPCR with increased stability in a particular conformationrelative to a second parent GPCR.

For the avoidance of doubt the first parent GPCR may be a GPCR which hasa naturally-occurring sequence, or it may be a truncated form or it maybe a fusion, either to the naturally-occurring protein or to a fragmentthereof, or it may contain mutations compared to the naturally-occurringsequence, providing that it retains ligand-binding ability.

Typically, identifying the position or positions at which the one ormore mutants have at least one different amino acid residue compared tothe first parent GPCR involves aligning their amino acid sequences withthat of the parent GPCR, for example using the Clustal W program(Thompson et al., 1994).

By “corresponding position or positions”, we include the meaning of theposition in the amino acid sequence of a second GPCR which aligns to theposition in the amino acid sequence of the first GPCR, when the firstand second GPCRs are compared by alignment, for example by usingMacVector and Clustal W. For example, as shown in the alignment in FIG.17, the six stabilising mutations in turkey β1-m23, R68S, M90V, Y227A,A282L, F327A and F338M, are at positions which correspond to residuesK60, M82, Y219, 0265, L310 and F321 respectively in the human β2receptor.

Having identified the corresponding position or positions in the aminoacid sequence of a second GPCR, the amino acids at those positions arereplaced with another amino acid. Typically, the amino acids arereplaced with the same amino acids which replaced the amino acids at thecorresponding positions in the mutant of the first parent GPCR (unlessthey are already that residue). For example, at position 68 in turkeyβ1-m23 (R68S), an arginine residue was replaced with a serine residue.Therefore, at the corresponding position in the human β2 receptor,position 60 (K60), the lysine residue is preferably replaced with aserine residue.

Mutations can be made in an amino acid sequence, for example, asdescribed above and using techniques well-established in the art.

It will be appreciated that the second GPCR may be any other GPCR. Forexample, stabilising mutations in a GPCR from one species may betransferred to a second GPCR from another species. Similarly,stabilising mutations in one particular GPCR isoform may be transferredto a second GPCR which is a different isoform. Preferably, the secondparent GPCR is of the same GPCR class or family as the first parentGPCR. Phylogenetic analyses have divided GPCRs into three main classesbased on protein sequence similarity, i.e., classes 1, 2, and 3 whoseprototypes are rhodopsin, the secretin receptor, and the metabotropicglutamate receptors, respectively (Foord et al (2005) Pharmacol. Rev.57, 279-288). Thus, the second GPCR may be a GPCR which is of the sameGPCR class as the first parent GPCR. Similarly, GPCRs have been dividedinto families by reference to natural ligands such as glutamate andGABA. Thus, the second GPCR may be of the same GPCR family as the firstparent GPCR. A list of GPCR classes and families has been produced bythe International Union of Pharmacology (Foord et al (2005) Pharmacol.Rev. 57, 279-288) and this list is periodically updated athttp://www.iuphar-db.org/GPCR/ReceptorFamilies Forward.

It will be appreciated that the second parent GPCR must be able to bealigned with the first parent GPCR such that the corresponding positionsof the mutations in the first GPCR can be determined in the second GPCR.Thus typically, the second parent GPCR has at least 20% sequenceidentity to the first parent GPCR and more preferably at least 30%, 40%,50%, 60%, 70%, 80% or 90% sequence identity to the first parent GPCR.However, some GPCRs have low sequence identity (e.g. family B and CGPCRs) and at the same time are very similar in structure. Thus the 20%sequence identity threshold is not absolute.

Method 4

The inventors have reasoned that the identification of structural motifsin which the one or more mutations in a mutant GPCR with increasedstability reside, will be useful in producing further mutant GPCRs withincreased stability.

Accordingly, a further method for producing a mutant G-protein coupledreceptor (GPCR) with increased stability relative to its parent GPCRcomprises:

-   -   a. providing one or more mutants of a first parent GPCR with        increased stability relative to the first parent GPCR    -   b. identifying in a structural membrane protein model the        structural motif or motifs in which the one or more mutants have        at least one different amino acid residue compared to the first        parent GPCR, and    -   c. making one or more mutations in the amino acid sequence that        defines a corresponding structural motif or motifs in a second        parent GPCR, to provide one or more mutants of a second parent        GPCR with increased stability relative to the second parent        GPCR.

Mapping stabilising mutations onto one or more known structural modelscan be used to identify particular structural motifs in which suchstabilising mutations reside. We have mapped stabilising mutations ofthe β1-adrenergic receptor onto structural models of the β2-adrenergicreceptor (Rasmussen et al (2007) Nature 450, 383-387; Cherezov et al(2007) Science 318:1258-65; Rosenbaum et al (2007) Science318:1266-1273) in order to identify such motifs. For example, Table (vi)lists the turkey β1-adrenergic receptor mutations which we have mappedonto the human β2-adrenergic receptor and describes the correspondingstructural motifs in which they reside. As discussed in Example 4,mapping of the Y227A mutation (equivalent to Y219 in the human β₂receptor) onto the human β₂-adrenergic receptor reveals its position atthe interface between helices such that the mutation may improve packingat the helical interface (see FIGS. 15, 16 and 23). Similarly, mappingof the M90V mutation (equivalent to M82 in the human β₂ receptor) ontothe human β₂-adrenergic receptor reveals it to be in helix 2 at a pointwhere the helix is kinked (see FIGS. 15, 16 and 20). Other mutationswere found to reside in further structural motifs includingtransmembrane helix surfaces pointing into the lipid bilayer,hydrophobic-hydrophilic boundary regions, protein binding pockets andloop regions (see Table (vi) and FIGS. 18-19, 21-22 and 24-25).

Such structural motifs, by virtue of them containing stabilisingmutations, are important in determining protein stability. Therefore,targeting mutations to these motifs will facilitate the generation ofstabilised mutant GPCRs. Indeed, there were several instances where morethan one mutation mapped to the same structural motif. For example, theY227A, V230A and A234L mutations in the turkey β1 adrenergic receptormapped to the same helical interface, the V89L and M90V mutations mappedto the same helical kink and the F327A and A334L mutations mapped to thesame helical surface pointing towards the lipid bilayer (Table (vi)).Thus, when one stabilising mutation has been identified, thedetermination of the structural motif in which that mutation is locatedwill enable the identification of further stabilising mutations.

The one or more mutants of a first parent GPCR may be selected orprepared according to any of the methods described above. Hence, thismethod may also be used to create stable, conformationally locked GPCRsby mutagenesis. For example, following the selection of mutant GPCRswhich have increased stability in a particular conformation, thestructural motifs in which such stabilising mutations reside can beidentified. Making one or more mutations in the amino acid sequence thatdefines the corresponding structural motif in another GPCR can then beused to produce a mutant GPCR with increased stability in a particularconformation relative to its parent GPCR.

We have performed a multiple sequence alignment of the human beta-2AR,rat NTR1, turkey beta-1 AR, human Adenosine A2aR and human muscarinic M1receptor amino acid sequences (FIG. 17) which shows that, when thethermostabilising mutations identified (see Examples 1-3) are positionedon the sequences then, in 11 instances out of a total of 70, twosequences contain mutations at the same position (denoted in FIG. 17with a star). Without wishing to be bound by any theory, the inventorsbelieve that thermostabilising mutations at these positions should be ofenhanced transferability for mapping onto a structural membrane proteinmodel. Thus in one embodiment, the mutant of the first parent GPCR is amutant human beta-2AR, rat NTR1, turkey beta-1 AR, human Adenosine A2aRor human muscarinic M1 receptor which, when compared to itscorresponding parent receptor, has a different amino acid at a positionwhich corresponds to any one or more of the following positionsaccording to the numbering of the human beta2 AR as set out in FIG. 17:Ala 59, Val 81, Ser 143, Lys 147, Val 152, Glu 180, Val 222, Ala 226,Ala 271, Leu 275 and Val 317.

In order to identify the structural motif or motifs, the stabilisingmutations are mapped onto a known structure of a membrane protein.

By “membrane protein” we mean a protein that is attached to orassociated with a membrane of a cell or organelle. Preferably, themembrane protein is an integral membrane protein that is permanentlyintegrated into the membrane and can only be removed using detergents,non-polar solvents or denaturing agents that physically disrupt thelipid bilayer.

The structural model of a membrane protein may be any suitablestructural model. For example, the model may be a known crystalstructure. Examples of GPCR crystal structures include bovine rhodopsin(Palczewski, K. et al., Science 289, 739-745. (2000)) and human β₂adrenergic receptor (Rasmussen at al, Nature 450, 383-7 (2007); Cherezovet al (2007) Science 318:1258-65; Rosenbaum et al (2007) Science318:1266-1273). The coordinates for the human β₂ adrenergic receptorstructure can be found in the RCSB Protein Data Bank under accessioncodes: 2rh1, 2r4r and 2r4s. Alternatively, the structural model may be acomputer generated model based upon homology or using de novo structureprediction methods (Qian at al Nature (2007) 450: 259-64).

It will be appreciated that stabilising mutations of a given mutant GPCRcan be mapped onto a structural model of any membrane protein which hassufficient structural similarity to the GPCR. In particular, the domainof the membrane protein must have sufficient structural similarity tothe GPCR domain in which the stabilising mutation resides, for a givenmutation to be transferable.

A protein domain is typically defined as a discretely folded assembly ofsecondary structure elements which may stand alone as a single proteinor be part of a larger protein in combination with other domains. It iscommonly a functional evolutionary unit.

GPCRs are essentially single domain proteins excluding those with largeN-terminal domains. Therefore, typically, the structural model is of amembrane protein which comprises at least one domain that has sufficientstructural similarity to the GPCR.

Structural similarity can be determined indirectly by the analysis ofsequence identity, or directly by comparison of structures.

With regard to sequence identity, the amino acid sequence encoding theGPCR domain in which the mutant has at least one different amino acidresidue compared to the first parent GPCR, is aligned with an amino acidsequence encoding a domain of a membrane protein for which a structuralmodel is available. It will be appreciated that one or more of thesesequences may contain an inserted sequence or N-terminal or C-terminalextensions which are additional to the core conserved domain. Foroptimal alignment, such sequences are removed so as not to skew theanalysis. Membrane proteins with sufficient sequence identity across thedomain in question may then be used as the structural model for mappingmutations. It has been shown for soluble protein domains that their 3Dstructure is broadly conserved above 20% sequence identity and wellconserved above 30% identity, with the level of structural conservationincreasing as sequence identity increases up to 100% (Ginalski, K. CurrOp Struc Biol (2006) 16, 172-177). Thus, it is preferred if thestructural membrane protein model is a model of a membrane protein whichcontains a domain that shares at least 20% sequence identity with themutant GPCR domain containing the at least one different amino acidresidue compared to the first parent GPCR, and more preferably at least30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity, and yet morepreferably at least 95% or 99% sequence identity.

Sequence identity may be measured by the use of algorithms such as BLASTor PSI-BLAST (Altschul et al, NAR (1997), 25, 3389-3402) or methodsbased on Hidden Markov Models (Eddy S et al, J Comput Biol (1995) Spring2 (1) 9-23). Typically, the percent sequence identity between twopolypeptides may be determined using any suitable computer program, forexample the GAP program of the University of Wisconsin Genetic ComputingGroup and it will be appreciated that percent identity is calculated inrelation to polypeptides whose sequence has been aligned optimally. Thealignment may alternatively be carried out using the Clustal W program(Thompson et al., 1994). The parameters used may be as follows: Fastpairwise alignment parameters: K-tuple(word) size; 1, window size; 5,gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.Multiple alignment parameters: gap open penalty; 10, gap extensionpenalty; 0.05. Scoring matrix: BLOSUM.

In addition to sequence identity, structural similarity can bedetermined directly by comparison of structural models. Structuralmodels may be used to detect regions of structural similarity notevident from sequence analysis alone, and which may or may not becontiguous in the sequence. For example, family B and C GPCRs arethought to share similar structures; however, their sequence identity isvery low. Similarly, the water transporting aquaporins spinach SoPip2,E. coli AqpZ, Methanococcus AqpM, rat Aqp4, human Aqp1 and sheep Aqp0share low sequence identity but all have similar structures.

Structural models of high fidelity may be constructed for proteins ofunknown structure using standard software packages such as MODELLER(Sali A and Blundell T, J Mol Biol (1993) 234(3) 779-815), wherein thestructure is modelled on a known structure of a homologous protein. Suchmodelling improves with increasing sequence identity. Typically, thesequence identity between the sequence of unknown structure and asequence of known 3D structure is more than 30% (Ginalski, K. Curr OpStruc Biol (2006) 16, 172-177). In addition, de novo structureprediction methods based on sequence alone may be used to model proteinsof unknown structure (Qian at al, (2007) Nature 450:259-64). Oncestructures have been experimentally determined or derived by modelling,regions of structural similarity may be detected by direct comparison oftwo or more 3D structures. They may, for example, comprise secondarystructure elements of a particular architecture and topology which canbe detected by the use of software such as DALI (Holm, L and Sander, C(1996) Science 273, 595-603). They may comprise local arrangements ofamino acid side chains and the polypeptide backbone, or specific sets ofatoms or groups of atoms in a particular spatial arrangement, which mayfor example also be detected by the use of graph theoreticalrepresentations (Artymiuk, P et al, (2005) J Amer Soc Info Sci Tech 56(5) 518-528). In this approach, the atoms or groups of atoms within theproteins or regions of proteins to be compared are typically representedas the nodes of a graph, with the edges of the graph describing theangles and distances between the nodes. Common patterns in these graphsindicate common structural motifs. This approach may be extended toinclude any descriptor of atoms or groups of atoms, such as hydrogenbond donor or acceptor, hydrophobicity, shape, charge or aromaticity;for example proteins may be spatially mapped according to suchdescriptors using GRID and this representation used as a basis forsimilarity searching (Baroni et al (2007) J Chem Inf Mod 47, 279-294).Descriptions of the methods, availability of software, and guidelinesfor user-defined selection of parameters, thresholds and tolerances aredescribed in the references given above.

In a preferred embodiment, the structural membrane protein model is astructural GPCR model. It will be appreciated that the structural modelof a GPCR may be a model of the first parent GPCR. For example,stabilising mutations within a mutant GPCR having increased stabilitycan be directly mapped onto the first parent GPCR structure and thestructural motifs in which such mutations are located, identified. Wherethe structure of the first parent GPCR is unknown, structural models ofother GPCRs may be used. For example, stabilising mutations in a GPCRfrom one species may be mapped onto a known structural model of the sameGPCR from another species. Similarly, stabilising mutations in oneparticular GPCR isoform may be mapped onto a known structural model ofanother GPCR isoform. Moreover, stabilising mutations from one GPCR maybe mapped onto a GPCR of the same class or family. A list of GPCRclasses and families has been produced by the International Union ofPharmacology (Foord et al (2005) Pharmacol. Rev. 57, 279-288) and thislist is periodically updated athttp://wvvw.iuphar-db.org/GPCR/ReceptorFamiliesForward.

As described above, it will be appreciated that the structural model maybe of any GPCR provided it has sufficient structural similarity acrossthe domain in which the mutant GPCR has at least one different aminoacid compared to the first parent GPCR. Thus, it is preferred if theGPCR shares at least 20% sequence identity with the mutant of the firstparent GPCR across the protein domain containing the at least onedifferent amino acid residue compared to the first parent GPCR, and morepreferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequenceidentity, and yet more preferably at least 95% or 99% sequence identity.However, the inventors recognise that the 20% sequence identitythreshold is not absolute. GPCRs with less than 20% sequence identity tothe first parent GPCR may also serve as a structural model to whichstabilising mutations are transferred, wherein the low sequence identityis counterbalanced by other similarities, including, for example, thepresence of the same sequence motifs, binding to the same G-protein orhaving the same function, or having substantially the same hydropathyplots compared to the first parent GPCR.

Mapping of stabilising mutations onto the structural model can be doneusing any suitable method known in the art. For example, typically, theamino acid sequence of the GPCR for which the structural model isavailable is aligned with the amino acid sequence of the mutant of thefirst parent GPCR. The position or positions of the at least onedifferent amino acid residue in the mutant GPCR relative to the firstparent GPCR can then be located in the amino acid sequence of the GPCRfor which a structural model is available.

By ‘structural motif’ we include the meaning of a three dimensionaldescription of the location in a GPCR structural model of athermostabilising mutation. For example, the structural motif may be anysecondary or tertiary structural motif within the GPCR. By ‘tertiarystructural motif’ we include any descriptor of atoms or groups of atoms,such as hydrogen bond donor or acceptor, hydrophobicity, shape, chargeor aromaticity. For example, proteins may be spatially mapped accordingto such descriptors using GRID and this representation used as a basisfor defining a structural motif (Baroni et al (2007) J Chem Inf Mod 47,279-294).

Table (vi) lists the structural motifs in which the turkey β1 adrenergicreceptor stabilising mutations were found to reside. As seen from thetable, the mutations are positioned in a number of distinct localities.Three mutations are in loop regions that are predicted to be accessibleto aqueous solvent. Eight mutations are in the transmembrane α-helicesand point into the lipid bilayer; three of these mutations are near theend of the helices and may be considered to be at thehydrophobic-hydrophilic boundary layer. Eight mutations are found at theinterfaces between transmembrane α-helices, three of which are eitherwithin a kinked or distorted region of the helix and another twomutations occur in one helix but are adjacent to one or more otherhelices which contain a kink adjacent in space to the mutated residue.These latter mutations could affect the packing of the amino acidswithin the kinked region, which could result in thermostabilisation.Another mutation is in a substrate binding pocket.

Accordingly, in one embodiment, the structural motif is any of a helicalinterface, a helix kink, a helix opposite a helix kink, a helix surfacepointing into the lipid bilayer, a helix surface pointing into the lipidbilayer at the hydrophobic-hydrophilic boundary layer, a loop region ora protein binding pocket.

Identifying a structural motif in which a stabilising mutation residessuggests the importance of that motif in protein stability. Therefore,making one or more mutations in the amino acid sequence that defines acorresponding structural motif or motifs in a second parent GPCR, shouldprovide one or more mutants of a second parent GPCR with increasedstability relative to the second parent GPCR.

The amino acid sequence which defines a structural motif is the primaryamino acid sequence of the amino acid residues which combine in thesecondary or tertiary structure of the protein to form the structuralmotif. It will be appreciated that such a primary amino acid sequencemay comprise contiguous or non-contiguous amino acid residues. Thus,identifying the amino acid sequence which defines the structural motifwill involve determining the residues involved and subsequently definingthe sequence. Mutations can be made in an amino acid sequence, forexample as described above and using techniques well-established in theart.

By “corresponding structural motif or motifs”, we mean the analogousstructural motif or motifs identified in the structural model which arepresent in the second parent GPCR. For example, if a helical interfacewas identified, the corresponding helical interface in the second parentGPCR would be the interface between the helices which are analogous tothe helices present in the structural model. If a helical kink wasidentified, the corresponding helical kink would be the kink in thehelix which is analogous to the kinked helix present in the structuralmodel. An analogous structural motif or motifs in the second parent GPCRcan be identified by searching for similar amino acid sequences in thesequence of the second parent GPCR which define the motif or motifs inthe structural model, for example, by sequence alignment. Moreover,computer based algorithms are widely available in the art that can beused to predict the presence of protein motifs based on an amino acidsequence. Thus, based upon the relative position of a particular motifwithin the amino acid sequence and its position relative to othermotifs, an analogous structural motif can readily be identified. It willbe appreciated that if a structural model of the second parent GPCR isavailable, the analogous structural motif or motifs can be directlymapped onto the structure of the protein. Typically, the amino acidsequence defining the analogous structural motif has at least 20%sequence identity with the sequence defining the motif in the structuralmodel, more preferably at least 30%, 40%, 50%, 60%, 70%, 80% and 90%sequence identity and yet more preferably 95% and 99% sequence identity.

In one embodiment, the second parent GPCR is the first parent GPCR. Forthe avoidance of doubt, the second parent GPCR may have thenaturally-occurring sequence of the first parent GPCR, or it may be atruncated form or it may be a fusion, either to the naturally occurringprotein or to a fragment thereof, or it may contain mutations comparedto the naturally-occurring sequence, providing that it retainsligand-binding.

In an alternative embodiment, the second parent GPCR is not the firstparent GPCR. For example, a mutant of a first parent GPCR may have beenidentified that has increased stability but it is desired to generate amutant of a different GPCR with increased stability. Preferably, thesecond parent GPCR is of the same GPCR class or family as the firstparent GPCR as described above. However, it will be appreciated that thesecond parent GPCR may be any known GPCR provided that it sharessufficient structural similarity with the first parent GPCR, such thatit contains a corresponding structural motif in which the stabilisingmutation of the mutant of the first parent GPCR resides. Thus typically,the second parent GPCR has at least 20% sequence identity to the firstparent GPCR and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or90% sequence identity. However, as mentioned above, some GPCRs have lowsequence identity (e.g. family B and C GPCRs) but are similar instructure. Thus the 20% sequence identity threshold is not absolute.

Since there are potentially thousands of mutations that can be screenedin a GPCR for increased stability, it is advantageous to targetparticular mutations which are known to be important in conferringstability. Therefore, it will be appreciated that the methods 3 and 4may be used in a method of selecting mutant GPCRs with increasedstability. In particular, carrying out methods 3 and 4 can be used totarget mutations to particular amino acid residues or to amino acidsequences which define structural motifs important in determiningstability.

Accordingly, in one embodiment, methods 3 and 4 further comprise:

-   -   (I) selecting a ligand, the ligand being one which binds to the        second parent GPCR when the GPCR is residing in a particular        conformation    -   (II) determining whether the or each mutant of the second parent        GPCR when residing in a particular conformation has increased        stability with respect to binding the selected ligand compared        to the stability of the second parent GPCR when residing in the        same particular conformation with respect to binding that        ligand, and    -   (III) selecting those mutants that have an increased stability        compared to the second parent GPCR with respect to binding the        selected ligand.

It will be noted that steps (I), (II) and (III) correspond to steps (b),(c) and (d) of method 1 described above. Accordingly, preferences forthe ligand and methods of assessing stability are as defined above withrespect to method 1.

Any mutant GPCR with increased stability relative to its parent GPCR,for example those provided by any of methods 1-4 described above, may beused in the present invention. For example, mutant GPCRs with increasedstability compared to their parent GPCRs, particularly those withincreased thermostability may be used. Particular examples of mutantGPCRs suitable for use in the present invention are provided below.

In one embodiment, the mutant GPCR is a mutant GPCR which has, comparedto its parent receptor, at least one different amino acid at a positionwhich corresponds to any one or more of the following positions: (i)according to the numbering of the turkey (3-adrenergic receptor as setout in FIG. 9: Ile 55, Gly 67, Arg 68, Val 89, Met 90, Gly 67, Ala 184,Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg 220, Ser 223,Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser 263, Leu 267,Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315, (iii) according tothe numbering of the rat neurotensin receptor as set out in FIG. 11: Ala69, Leu 72, Ala 73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109,Leu 111, Asp 113, Ile 116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165,Glu 166, Lys 176, Ala 177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189,Leu 205, Thr 207, Gly 209, Gly 215, Val 229, Met 250, Ile 253, Leu 256,Ile 260, Asn 262, Val 268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306,Leu 308, Val 309, Leu 310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351,Ala 356, Phe 358, Val 360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385,Cys 386, Pro 389, Gly 390, Trp 391, Arg 392, His 393, Arg 395, Lys 397,Pro 399, and (iv) according to the numbering of the muscarinic receptoras set out in FIG. 17: Leu 65, Met 145, Leu 399, Ile 383 and Met 384.

Alignment of the turkey β1 AR, human adenosine receptor, rat neurotensinreceptor and human muscarinic receptor amino acid sequences in FIG. 17,shows that in 11 instances out of 70, two sequences contain mutationsart the same position, namely at the following positions according tothe numbering of the human beta2 AR as set out in FIG. 17: Ala 59, Val81, Ser 143, Lys 147, Val 152, Glu 180, Val 222, Ala 226, Ala 271, Leu275 and Val 317. Therefore, in a further embodiment, the mutant GPCR isone which has, compared to its parent receptor, a different amino acidat any one of these positions.

Mutant β-Adrenergic Receptor

β-adrenergic receptors are well known in the art. They share sequencehomology to each other and bind to adrenalin.

In one embodiment, the mutant GPCR is a mutant β-adrenergic receptorwhich, when compared to the corresponding wild-type β-adrenergicreceptor, has a different amino acid at a position which corresponds toany one or more of the following positions according to the numbering ofthe turkey p-adrenergic receptor as set out in FIG. 9: Ile 55, Gly 67,Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe327, Ala 334, Phe 338.

The mutant β-adrenergic receptor may be a mutant of any β-adrenergicreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given turkey β-adrenergicreceptor amino acid sequence.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given turkeyβ-adrenergic receptor sequence, as determined using MacVector andCLUSTALW (Thompson et al (1994) Nucl. Acids Res. 22, 4673-4680). Morepreferably, the mutant receptor has at least 30% or at least 40% or atleast 50% amino acid sequence identity. There is generally a higherdegree of amino acid sequence identity which is conserved around theorthosteric (“active”) site to which the natural ligand binds.

As is described in Example 1 and FIG. 1 below, individual replacement ofthe following amino acid residues in the parent turkey β-adrenergicsequence (as shown in FIG. 9) lead to an increase in thermostability:Ile 55, Gly 67, Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val160, Gln 194, Gly 197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala282, Asp 322, Phe 327, Ala 334, Phe 338.

Thus, a mutant turkey β-adrenergic receptor in which, compared to itsparent, one or more of these amino acid residues have been replaced byanother amino acid residue may be used. Mutant β-adrenergic receptorsfrom other sources in which one or more corresponding amino acids in theparent receptor are replaced by another amino acid residue may also beused.

In one embodiment the mutant GPCR is a mutant Ji-adrenergic receptorwhich has at least one different amino acid residue in a structuralmotif in which the mutant receptor compared to its parent receptor has adifferent amino acid at a position which corresponds to any of thefollowing positions according to the numbering of the turkeyβ-adrenergic receptor as set out in FIG. 9: Ile 55, Gly 67, Arg 68, Val89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly 197, Leu221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala334, Phe 338.

For the avoidance of doubt, the parent may be a β-adrenergic receptorwhich has a naturally-occurring sequence, or it may be a truncated formor it may be a fusion, either to the naturally occurring protein or to afragment thereof, or it may contain mutations compared to thenaturally-occurring sequenced provided that it retains ligand-bindingability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another β-adrenergic receptor which aligns to thegiven amino acid residue in turkey β-adrenergic receptor when the turkeyβ-adrenergic receptor and the other β-adrenergic receptor are comparedusing MacVector and CLUSTALW.

FIG. 9 shows an alignment between turkey β-adrenergic receptor and humanβ1, β2 and β3 β-adrenergic receptors.

It can be seen that Ile 72 of human β1 corresponds to Ile 55 of turkeyβ-adrenergic receptor; Ile 47 of human β2 corresponds to Ile 55 ofturkey β-adrenergic receptor; and Thr51 of human β3 corresponds to Ile55 of turkey β-adrenergic receptor. Other corresponding amino acidresidues in human β1, β2 and β3 can readily be identified by referenceto FIG. 9.

It is preferred that the particular amino acid is replaced with an Ala.However, when the particular amino acid residue is an Ala, it ispreferred that it is replaced with a Leu (for example, see turkeyβ-adrenergic Ala 234, Ala 282 and Ala 334 in FIG. 1).

It is preferred if the mutant β-adrenergic receptor has a differentamino acid compared to its parent at more than one amino acid positionsince this is likely to give greater stability. Particularly preferredhuman β1 receptor mutants are those in which one or more of thefollowing amino acid residues are replaced with another amino acidresidue: K85, M107, Y244, A316, F361 and F372. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β1 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are preferred.

Particularly preferred human β2 receptor mutants are those in which oneor more of the following amino acids are replaced with another aminoacid residue: K60, M82, Y219, C265, L310 and F321. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β2 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are preferred.

FIG. 26 shows the effect on thermostability when six thermostabilisingmutations in β1-m23 (R68S, M90V, Y227A, A282L, F327A, F338M) weretransferred directly to the human β2 receptor (equivalent mutationsK60S, M82V, Y219A, C265L, L310A, F321M), making human β2-m23. The Tmsfor human β2 and β2-m23 were 29° C. and 41° C. respectively, thusexemplifying the transferability of thermostabilising mutations from onereceptor to another receptor. Accordingly, a particularly preferredhuman β2 receptor mutant is one which comprises the mutations K60S,M82V, Y219A, C265L, L310A, F321M.

Particularly preferred human β3 receptor mutants are those in which oneor more of the following amino acids are replaced with another aminoacid residue: W64, M86, Y224, P284, A330 and F341. Typically, the givenamino acid residue is replaced with Ala or Val or Met or Leu or Ile(unless they are already that residue).

Mutant human β3 receptors which have combinations of 3 or 4 or 5 or 6mutations as described above are preferred.

Particularly preferred combinations of mutations are described in detailin Tables 1 and 2 in Example 1, and suitable mutants include the mutantturkey β-adrenergic receptors, and also include mutant β-adrenergicreceptors where amino acids in corresponding position have been replacedby another amino acid, typically the same amino acid as indicated inTables 1 and 2 in Example 1.

Particularly preferred mutants are those which contain mutations in theamino acids which correspond to the given amino acid residue byreference to turkey β-adrenergic receptor: (R68S, Y227A, A282L, A334L)(see m6-10 in Table 2 below); (M90V, Y227A, F338M) (see m7-7 in Table 2below); (R68S, M90V, V230A, F327A, A334L) (see m10-8 in Table 2 below);and (R68S, M90V, Y227A, A282L, F327A, F338M) (see m23 in Table 2 below).

Mutant Adenosine Receptor

Adenosine receptors are well known in the art. They share sequencehomology to each other and bind to adenosine.

In one embodiment, the mutant GPCR is a mutant adenosine receptor which,when compared to the corresponding wild-type adenosine, has a differentamino acid at a position which corresponds to any one or more of thefollowing positions according to the numbering of the human adenosineA_(2a) receptor as set out in FIG. 10: Gly 114, Gly 118, Leu 167, Ala184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg 220, Ser223, Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser 263, Leu267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315, Ala 54, Val57, His 75, Thr 88, Gly 114, Gly 118, Thr 119, Lys 122, Gly 123, Pro149, Glu 151, Gly 152, Ala 203, Ala 204, Ala 231, Leu 235, Val 239.

The mutant adenosine receptor may be a mutant of any adenosine receptorprovided that it is mutated at one or more of the amino acid positionsas stated by reference to the given human adenosine A_(2a) receptoramino acid sequence.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given humanadenosine A_(2a) receptor sequence, as determined using MacVector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% or at least 60% sequence identity. Typically, there is ahigher degree of sequence conservation at the adenosine binding site.

As is described in Example 2 below, individual replacement of thefollowing amino acid residues in the human adenosine A_(2a) receptorsequence (as shown in FIG. 10) lead to an increase in thermostabilitywhen measured with the agonist 5′-N-ethylcarboxamidoadenosine (NECA):

Gly 114, Gly 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210,Ser 213, Glu 219, Arg 220, Ser 223, Thr 224, Gln 226, Lys 227, His 230,Leu 241, Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311,Pro 313, Lys 315.

Replacement of the following amino acid residues in the human A_(2a)receptor sequence (as shown in FIG. 10) lead to an increase inthermostability when measured with the antagonist ZM 241385(4-[2-[[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl]amino]ethyl]phenol):

Ala 54, Val 57, His 75, Thr 88, Gly 114, Gly 118, Thr 119, Lys 122, Gly123, Pro 149, Glu 151, Gly 152, Ala 203, Ala 204, Ala 231, Leu 235, Val239.

Thus, a mutant human adenosine A_(2a) receptor in which, compared to itsparent, one or more of these amino acid residues have been replaced byanother amino acid residue may be used. Mutant adenosine receptors fromother sources in which one or more corresponding amino acids in theparent receptor are replaced by another amino acid residue may also beused.

In one embodiment, the mutant GPCR is a mutant adenosine receptor whichhas at least one different amino acid residue in a structural motif inwhich the mutant receptor compared to its parent receptor has adifferent amino acid at a position which corresponds to any of thefollowing positions according to the numbering of the human adenosineA_(2a) receptor as set out in FIG. 10: Gly 114, Gly 118, Leu 167, Ala184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg 220, Ser223, Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser 263, Leu267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315.

For the avoidance of doubt, the parent may be an adenosine receptorwhich has a naturally-occurring sequence, or it may be a truncated formor it may be a fusion, either to the naturally-occurring protein or to afragment thereof, or it may contain mutations compared to thenaturally-occurring sequence, provided that it retains ligand-bindingability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another adenosine receptor which aligns to thegiven amino acid residue in human adenosine A_(2a) receptor when thehuman adenosine A_(2a) receptor and the other adenosine receptor arecompared using MacVector and CLUSTALW.

FIG. 10 shows an alignment between human adenosine A_(2a) receptor andthree other human adenosine receptors (A2b, A3 and A1).

It can be seen that, for example, Ser 115 in the A_(2b) receptor(indicated as AA2BR) corresponds to Gly 114 in the A_(2a) receptor.Similarly, it can be seen that Ala 60 in the A₃ receptor (indicated asAA3R) corresponds to Ala 54 in the A_(2a) receptor, and so on. Othercorresponding amino acid residues in human adenosine receptors A_(2b),A₃ and A₁ can readily be identified by reference to FIG. 10.

It is preferred that the particular amino acid in the parent is replacedwith an Ala. However, when the particular amino acid residue in theparent is an Ala, it is preferred that it is replaced with a Leu.

It is preferred that the mutant adenosine receptor has a different aminoacid compared to its parent at more than one amino acid position.Particularly preferred human adenosine A2b receptors are those in whichone or more of the following amino acid residues are replaced withanother amino acid residue: A55, T89, R123, L236 and V240. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue).

Mutant human adenosine A2b receptors which have combinations of 3 or 4or 5 mutations as described above are preferred.

Particularly preferred human adenosine A3 receptors are those in whichone or more of the following amino acid residues are replaced withanother amino acid residue: A60, T94, W128, L232 and L236. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue).

Mutant human adenosine A3 receptors which have combinations of 3 or 4 or5 mutations as described above are preferred.

Particular preferred human adenosine A1 receptors are those in which oneor more of the following residues are replaced: A57, T91, A125, L236,and L240. Typically, the given amino acid residue is replaced with Alaor Val or Met or Leu or Ile (unless they are already that residue).

Particularly preferred combinations of mutations are described in detailin Example 2. Suitable mutants include these mutant human adenosineA_(2a) receptors, and also include other mutant adenosine receptorswhere amino acids in corresponding positions have been replaced byanother amino acid, typically the same amino acid as indicated inExample 2.

Particularly preferred adenosine receptor mutants are those whichcontain mutations in the amino acids which correspond to the given aminoresidue by reference to human adenosine A2a receptor: (A54L, K122A,L235A) (Rant 17); (A54L, T88A, V239A, A204L) (Rant 19); and (A54L, T88A,V239A, K122A) (Rant 21).

Mutant Neurotensin Receptor

Neurotensin receptors are known in the art. They share sequence homologyand bind neurotensin.

In one embodiment, the mutant GPCR is a mutant neurotensin receptorwhich, when compared to the corresponding wild-type neurotensinreceptor, has a different amino acid at a position which corresponds toany one or more of the following positions according to the numbering ofthe rat neurotensin receptor as set out in FIG. 11: Ala 69, Leu 72, Ala73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp113, He 116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys176, Ala 177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr207, Gly 209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn262, Val 268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val309, Leu 310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe358, Val 360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro389, Gly 390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given ratneurotensin receptor sequence, as determined using MacVector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% amino acid sequence identity.

The mutant neurotensin receptor may be a mutant of any neurotensinreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given rat neurotensin receptoramino acid sequence.

As is described in Example 3 below, individual replacement of thefollowing amino acid residues in the rat neurotensin receptor sequence(as shown in FIGS. 11 and 28) lead to an increase in thermostabilitywhen considered with respect to the absence of neurotensin. Leu 72, Ala86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile116, Ala 120, Asp 139, Phe 147, Ala 155, Lys 176, Thr 179, Met 181, Ser182, Phe 189, Leu 205, Thr 207, Gly 209, Gly 215, Leu 256, Asn 262, Val268, Met 293, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Ser 362, Ala385, Cys 386, Trp 391, Arg 392, His 393, Lys 397, Pro 399.

As is described in Example 3 below, individual replacement of thefollowing amino acid residues in the rat neurotensin receptor sequence(as shown in FIGS. 11 and 28) lead to an increase in thermostabilitywhen considered with respect to the presence of neurotensin. Ala 69, Ala73, Ala 86, Ala 90, His 103, Val 165, Glu 166, Ala 177, Arg 183, Gly215, Val 229, Met 250, Ile 253, Ile 260, Thr 279, Thr 294, Gly 306, Leu308, Val 309, Leu 310, Val 313, Phe 342, Phe 358, Val 360, Ser 362, Asn370, Ser 373, Phe 380, Ala 385, Pro 389, Gly 390, Arg 395.

Thus, a mutant rat neurotensin receptor in which, compared to itsparent, one or more of these amino acid residues have been replaced byanother amino acid residue may be used. Mutant neurotensin receptorsfrom other sources in which one or more corresponding amino acids in theparent receptor are replaced by another amino acid residue may also beused.

In one embodiment the mutant GPCR is a mutant neurotensin receptor whichhas at least one different amino acid residue in a structural motif inwhich the mutant receptor compared to its parent receptor has adifferent amino acid at a position which corresponds to any of thefollowing positions according to the numbering of the rat neurotensinreceptor as set out in FIG. 11: Ala 69, Leu 72, Ala 73, Ala 86, Ala 90,Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile 116, Ala 120,Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys 176, Ala 177, Thr 179,Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, Gly 209, Gly 215,Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn 262, Val 268, Asn 270,Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val 309, Leu 310, Val 313,Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Val 360, Ser 362,Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro 389, Gly 390, Trp 391,Arg 392, His 393, Arg 395, Lys 397, Pro 399.

For the avoidance of doubt the parent may be a neurotensin receptorwhich has a naturally-occurring sequence, or it may be a truncated formor it may be a fusion, either to the naturally-occurring protein or to afragment thereof, or it may contain mutations compared to thenaturally-occurring sequence, providing that it retains ligand-bindingability.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another neurotensin receptor which aligns to thegiven amino acid residue in rat neurotensin receptor when the ratneurotensin receptor and the other neurotensin receptor are comparedusing MacVector and CLUSTALW.

FIG. 11 shows an alignment between rat neurotensin receptor and twohuman neurotensin receptors 1 and 2. It can be seen, for example, thatAla 85 of the human neurotensin receptor 1 corresponds to Ala 86 of therat neurotensin receptor, that Phe 353 of the human neurotensin receptor1 corresponds to Phe 358 of the rat neurotensin receptor, and so on.Other corresponding amino acid residue in the human neurotensinreceptors 1 and 2 can readily be identified by reference to FIG. 11.

It is preferred that the particular amino acid in the parent is replacedwith an Ala. However, when the particular amino acid residue in theparent is an Ala, it is preferred that it is replaced with a Leu.

It is preferred that the mutant neurotensin receptor has a differentamino acid compared to its parent at more than one amino acid position.Particularly preferred human neurotensin receptors (NTR1) are those inwhich one or more of the following amino acid residues are replaced withanother amino acid residue: Ala 85, His 102, Ile 259, Phe 337 and Phe353. Typically, the given amino acid residues is replaced with Ala orVal or Met or Leu or Ile (unless they are already that residue).

Mutant human neurotensin receptors (NTR1) which have combinations of 3or 4 or 5 mutations as described above are preferred.

Particularly preferred human neurotensin receptors (NTR2) are those inwhich one or more of the following amino acid residues are replaced withanother amino acid residue: V54, R69, T229, P331 and F347. Typically,the given amino acid residue is replaced with Ala or Val or Met or Leuor Ile (unless they are already that residue). Mutant human neurotensinreceptors (NTR2) which have combinations of 3 or 4 or 5 mutations asdescribed above are preferred.

Particularly preferred combinations of mutations are described in detailin Example 3. Suitable mutants include these mutant rat neurotensinreceptors, and also include other mutant neurotensin receptors whereamino acids in corresponding positions have been replaced by anotheramino acid, typically the same amino acid as indicated in Example 3.

Particularly preferred neurotensin receptor mutants are those whichcontain mutations in the amino acid residues which correspond to thegiven amino acid residue by reference to the rat neurotensin receptor:(F358A, A86L, I260A, F342A) (Nag7m); (F358A, H103A, 1260A, F342A)(Nag7n).

Mutant Muscarinic Receptor

Muscarinic receptors are known in the art. They share sequence homologyand bind muscarine.

In one embodiment, the mutant GPCR is a mutant muscarinic receptorwhich, when compared to the corresponding wild-type muscarinic receptor,has a different amino acid at a position which corresponds to any one ormore of the following positions according to the numbering of the humanmuscarinic receptor M1 as set out in FIG. 17: Leu 65, Met 145, Leu 399,Ile 383 and Met 384.

It is particularly preferred if the mutant GPCR is one which has atleast 20% amino acid sequence identity when compared to the given humanmuscarinic receptor sequence, as determined using MacVector andCLUSTALW. Preferably, the mutant GPCR has at least 30% or at least 40%or at least 50% amino acid sequence identity.

The mutant muscarinic receptor may be a mutant of any muscarinicreceptor provided that it is mutated at one or more of the amino acidpositions as stated by reference to the given muscarinic receptor aminoacid sequence.

Thus, a mutant human muscarinic receptor in which, compared to itsparent, one or more of these amino acid residues have been replaced byanother amino acid residue may be used. Mutant muscarinic receptors fromother sources in which one or more corresponding amino acids in theparent receptor are replaced by another amino acid residue may also beused.

For the avoidance of doubt the parent may be a muscarinic receptor whichhas a naturally-occurring sequence, or it may be a truncated form or itmay be a fusion, either to the naturally-occurring protein or to afragment thereof, or it may contain mutations compared to thenaturally-occurring sequence, providing that it retains ligand-bindingability.

In one embodiment, the mutant GPCR is a mutant muscarinic receptor. Forexample, the mutant muscarinic receptor may have at least one differentamino acid residue in a structural motif in which the mutant receptorcompared to its parent receptor has a different amino acid at a positionwhich corresponds to any of the following positions according to thenumbering of the human muscarinic receptor as set out in FIG. 17: Leu65, Met 145, Leu 399, Ile 383 and Met 384.

By “corresponding amino acid residue” we include the meaning of theamino acid residue in another muscarinic receptor which aligns to thegiven amino acid residue in human muscarinic receptor when the humanmuscarinic receptor and the other muscarinic receptor are compared usingMacVector and CLUSTALW.

It is preferred that the particular amino acid is replaced with an Ala.However, when the particular amino acid residue is an Ala, it ispreferred that it is replaced with a Leu.

It is preferred that the mutant GPCRs used in the invention haveincreased stability to any one of heat, a detergent, a chaotropic agentand an extreme of pH.

It is preferred if the mutant GPCRs used in the invention have increasedthermostability.

It is preferred that the mutant GPCRs used in the invention, includingthe mutant β-adrenergic, adenosine and neurotensin receptors, have anincreased thermostability compared to its parent when in the presence orabsence of a ligand thereto. Typically, the ligand is an antagonist, afull agonist, a partial agonist or an inverse agonist, whetherorthosteric or allosteric. As discussed above, the ligand may be apolypeptide, such as an antibody.

It is preferred that the mutant GPCRs used in the invention, for examplea mutant β-adrenergic receptor or a mutant adenosine receptor or amutant neurotensin receptor or a mutant muscarinic receptor is at least2° C. more stable than its parent preferably at least 5° C. more stable,more preferably at least 8° C. more stable and even more preferably atleast 10° C. or 15° C. or 20° C. more stable than its parent. Typically,thermostability of the parent and mutant receptors are measured underthe same conditions. Typically, thermostability is assayed under acondition in which the GPCR resides in a particular conformation.Typically, this selected condition is the presence of a ligand whichbinds the GPCR.

It is preferred that the mutant GPCRs used in the invention, whensolubilised and purified in a suitable detergent has a similarthermostability to bovine rhodopsin purified in dodecyl maltoside. It isparticularly preferred that the mutant GPCR retains at least 50% of itsligand binding activity after heating at 40° C. for 30 minutes. It isfurther preferred that the mutant GPCR retains at least 50% of itsligand binding activity after heating at 55° C. for 30 minutes.

For the avoidance of doubt, the mutant GPCR provided in step (a) of themethod of the first aspect of the invention may be extended ortruncated, contain internal deletions or insertions or otherwise alteredbeyond introduction of stabilising mutations; for example byintroduction or deletion of sites for posttranslational modificatione.g. glycosylation or phosphorylation or fatty acylation. It may also bechemically modified synthetically, for example by peptide semisynthesisor crosslinking or alkylation. In any event, the mutant GPCR providedhas increased stability in a particular conformation relative to itsparent GPCR.

Selection of Binding Partners

Selection for binding partners against membrane proteins has previouslyproven to be a difficult task. The preparation of the required pureantigen is problematic. Membrane proteins are oftendetergent-solubilised and they are purified as protein-detergentcomplexes. The type and concentration of detergent is crucial to keepthe protein in its native conformation. Some detergents may preventproteins from binding to plastic and polystyrene surfaces used as commonELISA supports. In addition, adsorption to the solid phase can causepartial denaturation of the protein.

In contrast, the increased stability of mutant GPCRs confers severaladvantages when the GPCRs are used as immunogens or selection reagentsfor screening, enabling them to be used as such in a variety ofcontexts.

Accordingly, in one embodiment, the mutant GPCR may be in a whole cellpreparation, a cell membrane fragment, solubilised in detergent or itmay be incorporated into a lipid monolayer, a lipid bilayer, abead-linked lipid particle, another solid-supported lipid layer or aproteoliposome. It is appreciated that when the GPCR is immobilised, thelipid layers may be supported directly as a layer on the surface of thesolid support or may be tethered as a layer or vesicle as described inCooper M. A. J Mol Recognit. 2004 July-August; 17(4):286-315.

The inventors recognise that high throughput membrane-receptor screeningis facilitated by immobilising membranes on beads or on surfaces thatcan be arrayed or otherwise multiplexed. Typically, membrane proteinsare deposited on a surface together with lipid in the form ofproteoliposomes. The most commonly employed methods for the formation ofproteoliposomes start with either prokaryote or eukaryote cells.Membrane proteins can be isolated either in a mixed micelle withdetergent, dissolved in an organic solvent or aggregated as membranefragments by sonication in buffer. Once isolated and purified, themembrane proteins can be reconstituted into vesicles by: a) organicsolvent-mediated reconstitutions (for example reverse-phase evaporation,rehydration of lipid-protein films), b) mechanical means (for examplesonication, French press, freeze-thaw), or c) detergent-mediated means(for example dialysis, dilution, or direct incorporation into pre-formedvesicles or into bicelles)

The detergent solubilised form of the mutant GPCR may be a partly pureor highly pure preparation. Purification, enabled by the improvedstability and optimisation of solubilisation conditions, confers theadvantage of removal of extraneous “sticky” antigens and lipids andother cell surface material such as carbohydrate to which, for example,phage may stick to. This is particularly beneficial when the‘extraneous’ material is charged or hydrophobic since phage particles,for example, are known to stick to hydrophobic or charged surfacesnon-specifically. Moreover, certain phage antibodies may themselves bindpromiscuously to antigens. A normal level of purity, as assessed bySDS-PAGE, is 80-95%. Therefore, preferably the detergent solubilisedform of the mutant GPCR is at least 80% pure, or at least 85% pure, andstill more preferably at least 90%, or at least 93%, or at least 95%,pure of other proteins. As is known in the art, higher levels of purity,e.g. at least 99%, can be achieved using additional purificationtechniques.

Solubilised receptor preparations are typically made in a buffer of 20mM Tris (pH 7.0), 0.1M (NH₄)₂SO₄, 10% glycerol, 0.07% CHS, 0.33% DOM,0.33% Chaps, 0.33 mM DOPC/DOPS (7:3), and 1 protease inhibitor tabletper 50 ml buffer. For solubilization trials, 0.5 ml of thissolubilization buffer is added to approximately 2×10⁶ cells, and thesecell suspensions are sonicated using a probe sonicator (6×1-s pulses)and placed on a rocker at 4° C. After 2 h, the solutions are centrifugedat 4° C. for 20 min at 14,000 rpm using a tabletop centrifuge. Thesupernatants containing solubilised receptor are then transferred to newtubes and kept frozen at −80° C. until analysis.

Preparations of mutant GPCRs formed from membrane fragments ormembrane-detergent extracts are reviewed in detail in Cooper M. A. J MolRecognit. 2004 July-August; 17(4):286-315, incorporated herein byreference. Of particular interest are methods adapted from Graham, J.M.; Higgins, J. A. Membrane Analysis; Springer-Verlag: New York, 1997and Dignam, J. D. Methods in Enzymology 1990, 182, 194-203. For example,one method is as follows; i) Spin 500 ml of a suitable cell culture(e.g. CHO, Sf9) at 1000 g for 10 min, re-suspend pellet in ca. twice thepellet volume of a suitable ice-cold 20 mM tris-HCl pH 8, 1 mM EDTA, 1mM EGTA, 0.1 mM PMSF, 2 μg/ml aprotinin, and 10 μg/ml leupeptin, ii)Homogenize with a Dounce homogenizer (type A) for 10 strokes, thencentrifuge at 30,000 g for 20 min, iii) Re-suspend pellet with 50 ml of20 mM tris HCl pH 8, 3 mM MgCl, 10 ug/ml DNAase I plus PMSF, 2 μg/mlaprotinin and 10 μg/ml leupeptin (solution B) and re-homogenize, iv)Centrifuge at 30,000 g for 20 min. and resuspend pellet in 20 ml ofsolution B and homogenize again, v) Store at 4° C. for immediate useafter snap freezing.

The mutant GPCR may be engineered to include a molecular tag at the Cterminus or N-terminus as is well known in the art. The tag may be anyof a FLAG tag, a His tag, a c-Myc tag, a DDDDK tag, an HSV tag, a Halotag or a biotin tag. Such tags can be used to facilitate phage-basedselection protocols in solution and may also be used to confer bindingto a solid support. Moreover, such tags will facilitate selection andenrichment of mutant GPCRs from impure preparations using affinitycolumns, affinity filters, magnetic beads and other examples ofselective solid supported reagents, filtration, centrifugation,size-exclusion chromatography and dialysis amongst other methods

The increased stability of mutant GPCRs in a range of detergents andsolubilisation buffers and additives lends them particularly well tobeing immobilised onto solid surfaces. Thus, in one embodiment themutant GPCR is immobilised onto a solid support. Various supports areknown in the art and include, for example, beads, columns, slides, chipsor plates. Immobilisation may be via covalent or non-covalentinteraction.

Where immobilisation is via a non-covalent interaction, the support maybe coated with any of avidin, streptavidin, a metal ion, an antibody tothe parent GPCR or an antibody to a molecular tag attached to the mutantGPCR. For example, the tag may be one recognised by an antibody such asa FLAG tag, or may be a poly-histidine tag enabling binding to a metalion such as nickel or cobalt, as described for example in Venturi et al,Biochemica et Biophysica Acta 1610 (2003) 46-50. Alternatively, themutant GPCR may be chemically modified for example with a biotin tagwhich can be bound to a surface coated with avidin or streptavidin.Moreover, a mutant GPCR may be immobilised via an antibody raised to thenative receptor sequence.

Where immobilisation is via a covalent interaction, the support may becoated with a polymeric support such as carboxylated dextran. Forexample, the mutant GPCR may be covalently immobilised onto a surfacecoated with a carboxylated polymer via amine coupling. For example,water-soluble carbodiimide mediated activation of a carboxymethylatedsupport such as dextran or hyaluronic acid allows for direct covalentcapture of a mutant GPCR via available amino moieties of the protein toform a stable amide linkage. Alternatively, GPCRs can be engineered orfurther derivatised with sulfydryl-reactive reagents (e.g.pyridinyldithioethanamine (PDEA) or 3-(2-pyridinyldithio)propioic acidN-hydroxysuccinimide ester) (SPDP)) which allows reaction with freesurface thiols (e.g. native free Cys, Met residues or with an engineeredC-terminal Cys residue) to form a reversible disulfide linkage. In asimilar manner, stable thioether bonds may be formed using maleimidecoupling reagents such assulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexanecarboxylate(Sulfo-SMCC) and N-(γ-maleimidobutyrloxy)sulfosuccinimide ester (GMBS).A solid support may also be derivatised with cystamine to effectcoupling with disulfide-activated GPCRs. Furthermore, treatment withhydrazine followed by a reductive amination enables coupling withaldheydes. The aldehyde groups may be native to the receptor or formedby mild oxidation of any cis-diols present in the solid support (presentin sugar residues of carbohydrates such as dextran, agarose, sepharose,hyaluronic acid and polyaginate). In addition to the above,amino-presenting surfaces can be treated with commercially availablebifunctional linking reagents to effect coupling with free amino orsulfhydryl groups on the receptor as described by (Ernst, O. P. et al.Meth. Enzymol. 2000, 315, 471-489 and Nunomura, W.e et al. J. Biol.Chem. 2000, 275, 24540-24546).

The orientation of the mutant GPCR will depend on the desired outcome ofthe binding partner identification. For example, for the identificationof therapeutic small molecules or antibodies, the mutant GPCR willtypically be immobilised via the C-terminus or other intracellulardomain to enable the extracellular domains to be outward facing in theassay. To identify native intracellular GPCR binding partners or agentswhich interfere with GPCR binding, the mutant GPCR is typicallyimmobilised by the N-terminus or extracellular domain. Immobilisation bythe N-terminus or extracellular domain may also identify therapeuticmolecules that bind to the intracellular domain. However, it will beappreciated that for such molecules to be active in vivo, they must beable to pass through the cell membrane.

In an alternative embodiment, the mutant GPCR is not immobilised and is,for example, solubilised in detergent or present in a whole cellpreparation. In this case, the test compound (for example, a library oftest compounds) may be immobilised on a solid support, such as a chipsurface. A variety of techniques are known in the art for immobilisingcompounds onto a chip surface, and any may be utilised. For example,suitable techniques include photolithography (Affymetrix, Santa Clara,Calif.), mechanical microspotting (Schena et al., Science (1995) vol.270, p. 467-470; Synteni, Fremont, Calif.) and ink jetting (IncytePharmaceuticals, Palo Alto, Calif.; and Protogene, Palo Alto, Calif.).The address of the test compound/GPCR mutant pair on the chip is used toreveal the identity of the test compound. Other suitable methods aredescribed, for example, in Fang, Y. et al. Drug Discovery Today 2003, 8,755-761, and references Alves, I. D. et al. Curr. Prot. & Peptide Sci.2005, 6, 293-312.; Barry, R. et al. Proteomics 2004, 4, 3717-3726.;Besenicar, M. et al. Chem. Phys. Lipids 2006, 141, 169-178.; Cocklin, S.et al. Prot. Sci. 2004, 13, 194-194.; Cooper, M. A. J. of Mol. Recognit.2004, 17, 286-315.; Fang, Y. et al., Drug Discov. Today 2003, 8,755-761.; Ferracci, G. et al. Anal. Biochem. 2004, 334, 367-375.;Graneli, A. et al. Anal. Biochem. 2007, 367, 87-94.; Graneli, A. et al.,Biosens. Bioelectron. 2004, 20, 498-504.; Groves, J. T. Curr. Op. DrugDiscov. Develop. 2002, 5, 606-612.; Groves, J. T. et al. J. Immunol.Meth. 2003, 278, 19-32.; Harding, P. J. et al. Eur. Biophys. J. Biophys.Let. 2006, 35, 709-712.; Komolov, K. E. et al. Anal. Chem. 2006, 78,1228-1234.; Kuroda, K. et al. App. Psychophys. Biofeedback 2006, 31,127-136.; Lang, M. J. et al. Curr. Prot. Peptide Sci. 2006, 7, 335-353.;Leifert, W. R. et al. J. Biomol. Screening 2005, 10, 765-779.;Martin-Garcia, J. et al. J. Virology 2005, 79, 6703-6713.; Minic, J. etal. Biochim. Biophys. Acta-General Subjects 2005, 1724, 324-332.;Mozsolits, H. et al. J. Peptide Sci. 2003, 9, 77-89.; Navratilova, I. etal. Anal. Biochem. 2006, 355, 132-139.; Ott, D. et al., Prot. Eng.Design & Selection 2005, 18, 153-160.; Park, P. S. H. et al. Febs Lett.2004, 567, 344-348.; Sobek, J. et al. Combinat. Chem. & High ThroughputScreening 2006, 9, 365-380.; Stenlund, P. et al. Analytical Biochemistry2003, 316, 243-250.; Winter, E. M. et al. Anal. Chem. 2006, 78,174-180.; Yokogawa, M. et al. J. Am. Chem. So. 2005, 127, 12021-12027.;Zurawski, J. A. et al. Biopolymers 2003, 71, 388-389, all of which areincorporated herein by reference.

In one embodiment, neither the GPCR nor the test compound areimmobilised, for example in phage-display selection protocols insolution.

The ability to produce milligram quantities of purified GPCRs,stabilised in a particular conformation, enables screening approacheswhich would not be available to native GPCRs present in biologicalmembranes. Thus, the method of the invention maybe used to identifyligands of ‘orphan’ GPCRs where the natural ligand is unknown. Ligandsof orphan GPCRs may be identified from biological samples such as bloodor tissue extracts or from libraries of ligands. Similarly, ligands ofmutant GPCRs may be identified where the ligands were interactingproteins such as accessory proteins. It is also appreciated that peptideor protein therapeutics may be identified by the methods of theinvention as could conformation-specific antibodies. For example,antibodies prepared as described below could be assessed forconformational-specific GPCR binding. In particular, antibodies may beidentified from supernatants obtained from B-cells taken from immunisedanimals, from hybridomas obtained following immortalisation of B-cellsfrom the immunised animal or from recombinant antibody libraries whichmay be expressed on phage particles or through an in vitro expressionsystem such as ribosome display. The methods of the invention may alsobe used to determine the mechanism of action of compounds where themechanism of action has not previously been known. For example, amixture of GPCRs representing the ‘GPCRome’ or a subset thereof could bescreened against compounds to identify if their mechanism of action wasvia binding to a GPCR. In addition, the invention may be used as abiochemical affinity purification system wherein particular compoundsare purified from a mixture of compounds.

The test compound may be provided as a biological sample. In particular,the sample could be any suitable sample taken from an individual. Forexample, the sample may be a fluid sample such as blood, serum, plasmaor spinal fluid. Alternatively, the sample could be a tissue or cellextract.

In one embodiment, the one or more test compounds is a polypeptide. Forexample, the test compound may be a particular type of polypeptide whichis known to bind to certain GPCRs but where the identification of aconformation-specific polypeptide is desired. Alternatively, thepolypeptide may be a candidate therapeutic molecule, for example ananticalin (Skerra J Biotechnol (2001) 74(4):257-75).

In one embodiment, the one or more test compounds is a peptide.

In one embodiment, the one or more test compounds is an affibody, apeptidomimetic, a nucleic acid, a peptide nucleic acid (PNA) or anaptamer, or a lipid or a carbohydrate.

In one embodiment, the one or more test compounds is a binding proteinbased on a modular framework, such as ankyrin repeat proteins, armadillorepeat proteins, leucine rich proteins, tetrariopeptide repeat proteinsor Designed Ankyrin Repeat Proteins (DARPins) or proteins based onlipocalin or fibronectin domains or Affilin scaffolds based on eitherhuman gamma crystalline or human ubiquitin.

In one embodiment, the one or more test compounds is a small molecule,for example a molecule less than 5000 daltons, or the one or more testcompounds is a natural product.

In one embodiment, the one or more test compounds is an antibody. Forexample, the test compound may be an antibody that has been raisedagainst a mutant GPCR of a parent GPCR, wherein the mutant GPCR hasincreased stability in a particular conformation relative to the parentGPCR. Preferably, the mutant GPCR is the same mutant GPCR as wasprovided in step (a).

As used herein, the term “antibody” includes but is not limited topolyclonal, monoclonal, chimaeric, single chain, Fab fragments andfragments produced by a Fab expression library. Such fragments includefragments of whole antibodies which retain their binding activity for atarget substance, Fv, F(ab′) and F(ab′)₂ fragments, as well asgenetically engineering derivatives of antibodies such as single chainantibodies (scFv), fusion proteins, domain antibodies (dAbs) anddiabodies. For example, it will be appreciated that recombinant DNAtechnology may be used to produce further antibodies or chimericmolecules which retain the binding specificity of an original antibody.Such technology may involve fusing the DNA encoding the immunoglobulinvariable region, or the complementarity determining regions (CDRs), ofan antibody to the constant regions, or constant regions plus frameworkregions of a different immunoglobulin, as described, for example, inEP-A-184187, GB 2188638A or EP-A-239400. Moreover, a hybridoma or othercell producing an antibody may be subject to genetic mutation or otherchanges which may or may not alter the binding specificity of antibodiesproduced. Thus, since antibodies can be modified in a number of ways,the term “antibody” is to be construed as covering any specific bindingmember or substance having a binding domain with the requiredspecificity. The term therefore includes antibody fragments,derivatives, functional equivalents and homologues of antibodies,including any polypeptide comprising an immunoglobulin binding domain,whether natural or wholly or partially synthetic. Chimeric moleculescomprising an immunoglobulin binding domain, or equivalent fused toanother polypeptide are therefore included. Furthermore, antibodies andfragments thereof may be human or humanised antibodies, as is well knownin the art.

Various procedures known within the art may be used to raise antibodiesagainst a mutant GPCR, or against fragments or fusions thereof.

For example, an antibody to a mutant GPCR having increased stabilityrelative to a parent GPCR may be produced by immunising a lymphocytewith an immunogen of the mutant GPCR, screening the antibodies soproduced for an antibody which binds to the GPCR, and isolating theantibody. By ‘immunising a lymphocyte’ we include both in vivoimmunisation, where a whole animal is immunised and in vitroimmunisation, where lymphocytes are immunised in vitro, for example asdescribed in U.S. Pat. No. 5,290,681.

Preferably, the immunogen of a mutant GPCR is generally all of themutant GPCR but may also be a part of the mutant GPCR, for example afragment of the mutant GPCR. For example, the production of a stabilisedGPCR may facilitate identification of a part of the GPCR not previouslyobvious from the native conformation that would benefit from a targetedimmunisation approach. The fragment of the mutant GPCR may be any partof the GPCR which is able to elicit an immune response such as anantibody response. It is known that peptides having as few as 5 aminoacids may elicit an antibody response, although typically largerpeptides are used. Thus, the fragment of the immunogen may have at least5 amino acids, typically from 5 to 1000 amino acids, such as 5 to 500, 5to 200, 5 to 100, 5 to 50, 5 to 40, 5 to 30, 5 to 20, for example 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

Furthermore, the immunogen may be a fusion of the mutant GPCR, whereinthe GPCR is fused to a second protein known to be immunogenic tolymphocytes. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. The immunogen may alsofurther include an adjuvant to enhance the immunological response to amutant GPCR in a mammal in vivo. Various adjuvants used to increase theimmunological response include, but are not limited to, Freund's(complete and incomplete), mineral gels (e.g., aluminum hydroxide),surface-active substances (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvantsusable in humans such as Bacille Calmette-Guerin and Corynebacteriumparvum, or similar immunostimulatory agents. An additional example of anadjuvant that can be employed includes MPL-TDM adjuvant (monophosphorylLipid A, synthetic trehalose dicorynomycolate). The choice of adjuvantcan be important in maintaining the structure of the GPCR and, for thisreason, Titremax and oil in water emulsions such as Ribi's adjuvantemulsion are particularly preferred. Stabilised mutant GPCRs may also belinked together on a backbone to produce polyvalent molecules to furtherincrease antigenicity.

It is appreciated that the immunogen of the mutant GPCR may be a variantof the mutant GPCR, provided that it is able to elicit an immuneresponse to the mutant GPCR and does not significantly reduce thestability of the mutant GPCR. Such variants include polypeptides whichhave one or more amino acid substitutions compared to the mutant GPCR,and as many as 5% substitutions. Typically, the substitutions areconservative substitutions where, for example, a “variant” refers to aprotein wherein at one or more positions there have been amino acidinsertions, deletions, or substitutions, either conservative ornon-conservative, provided that such changes result in a protein that isstill able to elicit an immune response against the mutant GPCR and doesnot significantly reduce the stability of the mutant GPCR. By“conservative substitutions” is intended combinations such as Gly, Ala;Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.Such variants of a mutant GPCR can be made using standard methods ofprotein engineering and site-directed mutagenesis.

It is appreciated that the immunogen may also be a polynucleotideencoding the stabilised GPCR or fragment thereof. For example, thepolynucleotide may be taken up into cells in vivo and expressed on thecell surface where it will stimulate an immune response.

The immunogen of a mutant GPCR may be provided as a whole cellpreparation, in a cell membrane fragment, solubilised in detergent, in alipid monolayer, in a lipid bilayer, in a bead-linked lipid particle, ina solid-supported lipid layer or in a proteoliposome, as describedabove.

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, chicken, mouse or other mammal) may beimmunized by one or more injections with the immunogen. The polyclonalantibody molecules directed against the immunogenic protein can beisolated from the mammal (e.g., from the serum or egg yolk) and furtherpurified by well known techniques, such as affinity chromatography usingprotein A or protein G, which provide primarily the IgG fraction ofimmune serum

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent.

Generally, either peripheral blood lymphocytes are used if cells ofhuman origin are desired, or spleen cells or lymph node cells are usedif non-human mammalian sources are desired. The lymphocytes are thenfused with an immortalized cell line using a suitable fusing agent, suchas polyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

Alternatively, monoclonal antibodies can be generated using the‘Selected Lymphocyte Antibody Method’ (SLAM) as is well known in the artand described, for example, in Babcook et al. (1996) Proc. Natl. Acad.Sci. 93:7843-7848. Briefly, a single lymphocyte that is producing anantibody with a desired specificity or function within a largepopulation of lymphoid cells is identified. Usually, lymphoid cellsderived from an in vivo immunization are screened for those that produceantibodies which bind to selected antigens using an adapted haemolyticplaque assay (Jerne & Nordin, 1963, Science, 140:405) and the geneticinformation that encodes the specificity of the antibody subsequentlyretrieved from that lymphocyte.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding monoclonalantibodies raised against mutant GPCRs with increased stability can bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells described above serve as a preferred source of such DNA.Once isolated, the DNA can be placed into expression vectors, which arethen transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also can be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

It will be appreciated that single-chain antibodies specific to GPCRscan also be produced (see e.g., U.S. Pat. No. 4,946,778). Antibodyfragments that contain the idiotypes to the mutant GPCR may also beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′2)) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′2)) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments. A general review of the techniques involved inthe synthesis of antibody fragments which retain their specific bindingsites is found in Winter & Milstein (1991) Nature 349, 293-299.

Bispecific antibodies may also be produced. Bispecific antibodies aremonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for a mutant GPCR. The secondbinding target is any other antigen, and advantageously is acell-surface protein or receptor, including another GPCR or receptorsubunit. For example, bispecific antibodies would be useful for pairs ofGPCRs which can form heterodimers (Milligan. Biochim Biophys Acta. 2007April; 1768(4):825-35.). In this case, the bispecific antibody mayselectively target specific heterodimeric receptors. Bispecificantibodies may also be useful for viral entry co-receptors where one ofthe co-receptors is a GPCR, e.g CD4 and the GPCRs CCR5 or CXCR4(Alkhatib G, Berger EA. Eur J Med Res. 2007 Oct. 15; 12(9):375-84).Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., 1991 EMBO J.,10:3655-3659.

It is appreciated that in some instances high throughput screening oftest compounds is preferred and that the method may be used as a“library screening” method, a term well known to those skilled in theart. Thus, the test compound may be a library of test compounds. Forexample, the library may be a peptide or protein library produced, forexample, by ribosome display or an antibody library prepared either invivo, ex vivo or in vitro. Methodologies for preparing and screeningsuch libraries are known in the art.

Thus, rather than the test compound being an antibody raised against amutant GPCR with increased stability in a particular conformationrelative to its parent GPCR and then testing its binding to that GPCR,the test compound may be an antibody library. Thus another method forgenerating antibodies specific to a GPCR involves screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria, yeast, filamentous phages, ribosomes or ribosomal subunitsor other display systems. In this method, large libraries of antibodysequences or antibody fragment sequences are obtained from diversesources such healthy donors, patients or animals (healthy or not). Thesesequences are cloned and expressed in an appropriate system andantibodies typically selected by binding to a GPCR with increasedstability immobilised on a solid surface.

A particular example of an antibody library is a recombinantcombinatorial antibody library, for example a scFv or Fab phage displaylibrary, prepared using human VL and VH cDNAs prepared from mRNA derivedfrom human lymphocytes (McCafferty et al., Nature 348:552-553 (1990)).According to this technique, antibody V domain genes are cloned in-frameinto either a major or minor coat protein gene of a filamentousbacteriophage, such as M 13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the ability of the antibody to bind to the GPCR withincreased stability also result in selection of the gene encoding theantibody exhibiting those properties. Phage display can be performed ina variety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-57 1(1993). Moreover, examples of methods and reagents particularly amenablefor use in generating and screening antibody display libraries can befound in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang etal. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et alPCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J. Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

The methods may also be used to identify a polynucleotide capable ofexpressing a polypeptide binding partner of a GPCR, for example asdescribed above in relation to phage display methods. Aliquots of anexpression library in a suitable vector may be tested for the ability togive the required result. It will be appreciated that several cycles ofidentifying pools of polynucleotides comprising a polynucleotide havingthe required property and then rescreening those polynucleotides may berequired in order to identify a single species of polynucleotide withthe required property.

The invention includes screening methods to identify drugs or leadcompounds of use in treating a disease or condition. It is appreciatedthat screening assays which are capable of high throughput operation areparticularly preferred.

It is appreciated that in the methods described herein, which may bedrug screening methods, a term well known to those skilled in the art,the test compound may be a drug-like compound or lead compound for thedevelopment of a drug-like compound.

The term “drug-like compound” is well known to those skilled in the art,and may include the meaning of a compound that has characteristics thatmay make it suitable for use in medicine, for example as the activeingredient in a medicament. Thus, for example, a drug-like compound maybe a molecule that may be synthesised by the techniques of organicchemistry, less preferably by techniques of molecular biology orbiochemistry, and is preferably a small molecule, which may be of lessthan 5000 daltons and which may be water-soluble. A drug-like compoundmay additionally exhibit features of selective interaction with aparticular protein or proteins and be bioavailable and/or able topenetrate target cellular membranes or the blood:brain barrier, but itwill be appreciated that these features are not essential.

The term “lead compound” is similarly well known to those skilled in theart, and may include the meaning that the compound, whilst not itselfsuitable for use as a drug (for example because it is only weakly potentagainst its intended target, non-selective in its action, unstable,poorly soluble, difficult to synthesise or has poor bioavailability) mayprovide a starting-point for the design of other compounds that may havemore desirable characteristics.

Thus in one embodiment, the method further comprises modifying a testcompound which has been shown to bind to the mutant GPCR when residingin a particular conformation, and determining whether the modified testcompound binds to the mutant GPCR when residing in the particularconformation. It will be appreciated that it may be further determinedwhether the modified test compound also binds to the parent GPCR whenresiding in the particular conformation.

Various methods may be used to determine binding between a GPCR and atest compound including, for example, enzyme linked immunosorbent assays(ELISA), surface plasmon resonance assays, chip-based assays,immunocytofluorescence, yeast two-hybrid technology and phage displaywhich are common practice in the art and are described, for example, inPlant et al (1995) Analyt Biochem, 226(2), 342-348. and Sambrook et al(2001) Molecular Cloning A Laboratory Manual. Third Edition. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. Other methods ofdetecting binding between a test compound and the GPCR includeultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods. Fluorescence Energy Resonance Transfer(FRET) methods, for example, well known to those skilled in the art, maybe used, in which binding of two fluorescent labelled entities may bemeasured by measuring the interaction of the fluorescent labels when inclose proximity to each other.

Where the mutant GPCR is provided as a whole cell preparation, amembrane protoplast or a proteoliposome, it will be appreciated thatbiophysical techniques such as patch clamping, magic angle spinning NMR,fluorescence correlation spectroscopy, fluorescence resonance energytransfer and analytical ultracentrifugation may be used to analysebinding of the mutant GPCR to the test compound (as described in New, R.C., Liposomes: a practical approach. 1st ed.; Oxford University Press:Oxford, 1990, and Graham, J. M.; Higgins, J. A., Membrane Analysis.Springer-Verlag: New York, 1997.) Methods which allow quantitative,non-invasive determination of both the affinity and kinetics of suchinteractions include direct assays that allow continuous monitoring ofmembrane-protein binding, or in some cases by ultra-rapid separation ofbound and free interactants followed by quantification of themembrane-bound and membrane-free analyte.

It will be appreciated that a bound test compound can be detected usinga unique label or tag associated with the compound such as a peptidelabel, a nucleic acid label (Kerr et al., JACS (1993) vol. 115, p.2529-2531; and Brenner & Lerner, Proc. Natl. Acad. Sci. USA (1992) vol.89, p. 5381-5383), a chemical label (Ohlmeyer et al., Proc. Natl. Acad.Sci. USA (1993) vol. 90, p. 109222-10926; and Maclean et al., Proc.Natl. Acad. Sci. USA (1997) vol. 94, p. 2805-2810); a fluorescent label(Yamashita & Weinstock (SmithKline Beecham Corporation), WO95/32425(1995); and Sebestyen et al., Pept. Proc. Eur. Pept. Symp. 22nd 1992(1993), p. 63-64), or a radio frequency tag (Nicolaou et al., Angew.Chem. Int. Ed. Engl. (1995) vol. 34, p. 2289-2291; and Moran et al.,JACS (1995) vol. 117, p. 10787-10788).

Where the test compound is an antibody against a mutant GPCR withincreased stability, produced using any of the methods described above,binding is preferably assayed using immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. For example, in an ELISA, typically, the stabilised GPCR isimmobilised on a microtitre plate or other solid surface. The plates arefirst incubated with BSA or other similar protein to block non-specificbinding sites. Samples containing the antibody (such as serum or eggyolk or hybridoma cell culture supernatant) are then added to the plateand the antibodies allowed to bind to the immobilised GPCR. Boundantibodies are detected by the addition of a second detection antibodywhich binds to the first antibody and allows detection via itsconjugation to an enzyme which catalyzes a reaction which can besubsequently detected. In addition, the binding affinity of antibodiescan also be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem, 107:220 (1980). Preferably, antibodies having a highdegree of specificity and a high binding affinity for the target GPCRare isolated.

It will be appreciated that where the antibody is a monoclonal antibodyderived from a hybridoma, the hybridoma clones identified as expressingspecific antibody can subsequently be subcloned by limiting dilutionprocedures and grown using standard methods. Suitable culture media forthis purpose include, for example, Dulbecco's Modified Eagle's Mediumand RPMI-1640 medium. Alternatively, the hybridoma cells can be grown invivo as ascites in a mammal. The monoclonal antibodies secreted by thesubclones can then be isolated or further purified from the culturemedium or ascites fluid by conventional immunoglobulin purificationprocedures such as, for example, protein A-Sepharose, hydroxylapatitechromatography, gel electrophoresis, dialysis, or affinitychromatography.

Where the test compound is a phage display antibody library, binding toa stabilised GPCR may be assessed as follows. Typically, the mutant GPCRwith increased stability is coated onto wells of a microtiter plateovernight at 4° C. The wells are washed in PBS and blocked for 1 hour at37° C. in MPBS (3% milk powder in PBS). Purified phage from a phagemidlibrary e.g. expressing a repertoire of human scFv (10 transducing units(tu)) are then blocked for 1 hour in a final volume of 100 μl of 3%MPBS. The blocked phage are added to the blocked GPCR wells andincubated for 1 hour. Wells are washed 5 times with PBST (PBS containing0.1% v/v Tween 20) before being wash 5 times with PBS. The bound phageparticles are then eluted and used to infect 10 ml exponentially growingE. coli TG1. The infected cells are grown in 2TY broth for 1 hour at 37°C., then spread onto 2TYAG plates and incubated overnight at 30° C.Cultures from this first round of panning selection are superinfectedwith helper phage and rescued to give, for example, scFvantibody-expressing phage particles for the second round of panning.

An alternative binding assay for phage display antibodies includes theuse of soluble selections using biotinylated mutant GPCR protein at afinal concentration typically of 100 nM. In this case, purified scFvphage (1012 tu) from a scFv phagemid library (as described above) issuspended in 1 ml 3% MPBS and blocked for 30 minutes. The biotinylatedGPCR is then added and incubated at room temperature for 1 hour. Thephage/antigen complexes are subsequently added to 250 μl of Dynal M280Streptavidin magnetic beads that have been blocked for 1 hour at 37° C.in 1 ml of 3% MPBS, and incubated with the beads for a further 15minutes at room temperature. The beads are captured using a magneticrack and washed 4 times in 1 ml of 3% MPBS/0.1% (v/v) Tween 20 followedby 3 washes in PBS. After the last PBS wash, beads are resuspended in100 μl PBS and used to infect 5 ml exponentially growing E. coli. Again,cultures from this first round of soluble selection would besuperinfected with helper phage and rescued to give scFvantibody-expressing phage particles for a second round of solubleselection.

It is appreciated that screening assays which are capable of highthroughput operation are particularly preferred to determine binding toa mutant GPCR, for example chip-based assays. Stabilised mutant GPCRsare particularly suited to such assays unlike their parent GPCRs whichare not stable enough when purified to be used in these formats. Inparticular, technology called VLSIPS™ has enabled the production ofextremely small chips that contain hundreds of thousands or more ofdifferent molecular probes, i.e. the test compounds. These biologicalchips have probes arranged in arrays, each probe assigned a specificlocation. Biological chips have been produced in which each location hasa scale of, for example, ten microns. The chips can be used to determinewhether target molecules interact with any of the probes on the chip.After exposing the array to target molecules under selected testconditions, scanning devices can examine each location in the array anddetermine whether a target molecule has interacted with the probe atthat location.

A test compound to a mutant GPCR on a chip surface may be detected byscanning the chip surface for radioactivity or fluorescence. The addressof the interacting pair on the chip reveals the identity of the testcompound or where there is an array of mutant GPCRs on the chip, theidentity of the receptor (see, for example, Kuimelis et al., AddressableProtein Arrays, U.S. Ser. No. 60/080,686, Apr. 3, 1998, now abandoned,and U.S. Ser. No. 09/282,734, Mar. 31, 1999). In the latter case thearray of mutant GPCRs may be used as a method for obtaining selectivitydata on compounds either for various conformations of the same GPCR orfor various GPCRs.

Alternative methods of detecting binding of a mutant GPCR to a testcompound, for example DNA, RNA, proteins and phospholipids, smallmolecules and natural products include surface plasmon resonance assays(SPA), for example as described in Plant et al (2005) Analyt Biochem226(2), 342-348. The mutant GPCR, immobilised on a SPA bead, may beincubated with a single ligand which is labelled for example with afluorescent group or the ligand may be a radioligand. The ability of atest compound to bind to the mutant GPCR may then be determined via itsability to displace the fluorescent ligand or the radioligand. Inanother example, the mutant GPCR is immobilised on a chip surface andbinding of test compounds is detected by surface plasmon resonance andrelated techniques employing evanescent waves. Changes in refractiveindex can be used to determine the amount of bound compound, theaffinity of interaction and the association and dissociation kinetics.An example of this approach has been described for rhodopsin which wasimmobilised on carboxylated dextran surfaces modified with long alkylgroups. Following amine coupling of the detergent-solubilised receptor,lipid/detergent-mixed micelles were adhered over the immobilizedsurface. The detergent was eluted in the subsequent buffer flow and theremaining lipid formed a bilayer on the chip surface. (Karlsson O P,Lofas S. Anal Biochem. 2002 Jan. 15; 300(2):132-8.

Where the test compound is a peptide or protein, for example, a ligandfor an orphan receptor or an interacting protein, the bound ligand maybe eluted and then identified by mass spectrometry such as matrixassisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF) or electrospray ionization mass spectrometry (ESI-MS)(Williams C. Addona T A. Trends Biotechnol. 2000 February; 18(2):45,Williams C Curr Opin Biotechnol. 2000 February; 11(1):42-6). The mutantGPCR may be immobilised on a column or bead, or expressed in tagged formin a cell and co-purified with the ligand from such complex mixturesusing reagents directed to the tag or directly to the GPCR (Rigaut G,Shevchenko A, Rutz B, Wilm M, Mann M, Séraphin B. Nat Biotechnol. 1999October; 17(10): 1030-2).

The ability to generate high affinity conformation specific bindingpartners to GPCRs will facilitate the production of therapeutic GPCRbinding partners. Thus, it will be appreciated that in addition toestablishing binding to a GPCR, it will also be desirable to determinethe functional effect of a binding partner on the GPCR.

Accordingly, in an embodiment of the invention, the method furthercomprises determining if the binding partner affects the function of theGPCR to which it binds and isolating a test compound that affects thefunction of the GPCR.

For example, in one embodiment, it is determined whether the bindingpartner alters the binding of the GPCR to its ligand. By ligand, weinclude any molecule which binds to the GPCR and which causes the GPCRto reside in a particular conformation as described above. Preferably,the ligand is the natural ligand of that GPCR or an analogue thereof.Binding of a GPCR to its ligand can be assayed using standard ligandbinding methods known in the art and, for example, as described above.For example, the ligand may be radiolabelled or fluorescently labelled.The binding assay can be performed using the stabilised mutant GPCR orthe parent GPCR. Typically, the stabilised GPCR is purified or expressedin a cell such a mammalian, bacterial or insect cell. Typically, theparent receptor is expressed in a cell such as a mammalian, bacterial orinsect cell. The assay may be carried out on whole cells or on membranesobtained from the cells. The binding partner will be characterised byits ability to alter the binding of the labelled ligand.

In one embodiment, the binding partner decreases binding between theGPCR and its ligand. For example, the binding partner may decreasebinding by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold,15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold or 1000 fold.Preferably, the binding partner decreases binding by a factor of between100-1000 fold, such as between 10-100 fold.

In one embodiment, the binding partner increases binding between theGPCR and its ligand. For example, the binding partner may increasebinding by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold,15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold or 1000 fold.Preferably, the binding partner increases binding by a factor of between100-1000 fold, such as between 10-100 fold.

In a further embodiment, it is determined whether the binding partnermodulates activation of a GPCR. For example, if a mutant GPCR stabilisedin an agonist conformation was provided in step (a) of the method of theinvention, the binding partner selected may be an agonist bindingpartner and thus increase activation of the GPCR. If a mutant GPCRstabilised in an antagonist conformation was provided in step (a) of themethod of the invention, the binding partner selected may be anantagonist binding partner and thus decrease activation of the GPCR.

In this assay the parent GPCR or stabilised mutant GPCR is expressed invivo, for example, in mammalian or insect cells where the GPCR isallowed to couple to well know GPCR signal transduction pathways (EglenR. M. Functional G protein-coupled receptor assays for primary andsecondary screening. Comb Chem High Throughput Screen. 2005 June;8(4):311-8). Such assays include: calcium mobilisation (Gonzalez J E,Maher M P. Cellular fluorescent indicators and voltage/ion probe reader(VIPR) tools for ion channel and receptor drug discovery. ReceptorsChannels. 2002; 8(5-6):283-95, Dupriez V J, Maes K, Le Poul E, BurgeonE, Detheux M. Aequorin-based functional assays for G-protein-coupledreceptors, ion channels, and tyrosine kinase receptors. ReceptorsChannels. 2002; 8(5-6):319-30), changes in cAMP levels (Weber M, FerrerM, Zheng W, Inglese J, Strulovici B, Kunapuli P. A 1536-well cAMP assayfor Gs- and Gi-coupled receptors using enzyme fragmentationcomplementation. Assay Drug Dev Technol. 2004 February; 2(1):39-49.),activation of kinase pathways (Leroy D, Missotten M, Waltzinger C,Martin T, Scheer A. G protein-coupled receptor-mediated ERK1/2phosphorylation: towards a generic sensor of GPCR activation. J ReceptSignal Transduct Res. 2007; 27(1):83-97), regulation of genetranscription for example via the use of a reporter gene (Liu B, Wu D.Analysis of the coupling of G12/13 to G protein-coupled receptors usinga luciferase reporter assay. Methods Mol Biol. 2004; 237:145-9, Kent TC, Thompson K S, Naylor L H. Development of a generic dual-reporter geneassay for screening G-protein-coupled receptors J Biomol Screen. 2005August; 10(5):437-46), recruitment of β-arrestin (Hudson C C, Oakley RH, Sjaastad M D, Loomis C R. High-content screening of known Gprotein-coupled receptors by arrestin translocation Methods Enzymol.2006; 414:63-78), activation of G proteins such as measuring GTPaseactivity (Jameson E E, Roof R A, Whorton M R, Mosberg H I, Sunahara R K,Neubig R R, Kennedy R T. Real-time detection of basal and stimulated Gprotein GTPase activity using fluorescent GTP analogues. J Biol Chem.2005 Mar. 4; 280(9):7712-9) or measuring [35S]GTPgamma(γ)S binding(Rodgers G, Hubert C, McKinzie J, Suter T, Statnick M, Emmerson P,Stancato L. Development of displacement binding and GTPgammaSscintillation proximity assays for the identification of antagonists ofthe micro-opioid receptor. Assay Drug Dev Technol. 2003 October;1(5):627-36).

Binding partners are typically selected which modulate the activation ofthe receptor.

For agonist binding partners the binding partner will typically mimicthe activity of the natural ligand of the receptor and produce anincrease in receptor activation, G protein activation or signaltransduction. This will occur in the absence of an additional agonist.An agonist binding partner may increase receptor activation by a factorof at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold,50 fold, 100 fold, 250 fold, 500 fold, 1000 fold, or 10000 fold.

It will be appreciated that there are two ways in which a bindingpartner may increase receptor activation. For example, the bindingpartner may act as a direct agonist, in which case receptor activationis typically increased by between 2-1000 fold. In another method, thebinding partner may act to amplify the activity of an agonist. Forexample, the binding partner may increase the potency of the agonist, inwhich case receptor activation is typically increased by between 2-1000fold, such as between 10-100 fold, or the binding partner may increasethe maximal response produced by the agonist, in which case receptoractivation is typically increased by between 2-10 fold. It will beappreciated that activating/amplifying the activity of receptors thatare already switched on by endogenous ligand may be preferable toturning on all available receptors, since it is more physiologicallyspecific and may mitigate issues such as desensitisation and undesirableside-effects (Christopoulos A (2002) Nat Rev Drug Discov 1:198-210).

In the case of antagonist binding partners the binding partner willtypically act to block the activity of the receptor or its activation byan agonist. The antagonist binding partner may do this by blocking thebinding of the agonist or by locking the receptor in an inactive formsuch that it is unable to couple to G proteins. An antagonist bindingpartner may decrease receptor activation by a factor of at least 2 fold,3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold, 50 fold, 100 fold,250 fold, 500 fold, 1000 fold or 10000 fold. Typically, an antagonistbinding partner would reduce receptor activation to a non-detectablelevel.

It will be appreciated that it may be desirable to produce a testcompound or combination of test compounds that bind to more than oneGPCR. For example, the test compound may bind to more than one GPCR ofthe same class of family. A list of GPCR classes and families has beenproduced by the International Union of Pharmacology (Foord et al (2005)Pharmacol. Rev. 57, 279-288) and this list is periodically updated athttp://www.iuphar-db.org/GPCR/ReceptorFamiliesForward. The test compoundmay bind to one target GPCR and at least one further GPCR. The at leastone further GPCR may be a GPCR that has been implicated in a diseasepathway, or a GPCR that is involved in the same signalling pathway asthe target GPCR, for example a signalling pathway that regulates ormodulates a disease pathway. In addition, the at least one further GPCRmay be one which enhances or suppresses the action of a test compound onthe target GPCR. Such enhancement or suppression of action can bedetermined using methods well known in the art including binding assaysand functional assays as described, for example, above.

Accordingly, in one embodiment more than one mutant GPCR is provided instep (a). For example, at least 2, 3, 4 or 5 mutant GPCRs of a differentparent GPCR may be provided in step (a). Thus, in this embodiment, testcompounds are selected for which bind to more than one GPCR. Thecompound may be a cross-reactive compound including, for example, asmall molecule, affibody, antibody or diabody. It will be appreciatedthat such an approach may lead to improvements in a binding partner'sefficacy or potency.

Thus, a test compound may be selected which binds to a first and secondGPCR, where the first and second GPCRs may be any pair of GPCRs. Forexample, the method can be used to select for a bivalent partner thatbinds to GPCRs which form a heterodimer, in which case the bivalentpartner could bind to both receptors at the same time. Examples ofappropriate GPCR heterodimers in this context include dopamine D1 andadenosine A1, opioid receptor heterodimers, cannabinoid CB1 and orexinreceptors (Marshall F H. Heterodimerization of G-protein-coupledreceptors in the CNS. Curr Opin Pharmacol. 2001 February; 1(1):40-4;Kent T, McAlpine C, Sabetnia S, Presland J. G-protein-coupled receptorheterodimerization: assay technologies to clinical significance. CurrOpin Drug Discov Devel. 2007 September; 10(5):580-9). Moreover, abinding partner could be selected for which binds to and modulates theactivity of two independent receptors for the purpose of enhancedtherapeutic activity. For example, CCK1/opioid receptor peptides bind toboth CCK1 and the opoid receptor (Garcia-López M T, González-Muñiz R,Martin-Martínez M, Herranz R. Strategies for design of non peptide CCK1Ragonist/antagonist ligands. Curr Top Med Chem. 2007; 7(12):1180-94).Other appropriate examples include a combined beta 2 agonist andmuscarinic antagonist; a dopamine D2 antagonist and 5HT2 antagonist; aD2 antagonist and 5HT6 antagonist; and a M1 agonist and 5HT6 antagonist.

It will be appreciated that the more than one GPCR provided in step (a)may or may not reside in the same conformation. For example, a diabodyor similar bivalent binding partner may agonise at one end of themolecule and antagonise at another end, in which case the GPCRs wouldnot have to reside in the same conformation.

Typically, where a test compound is selected that binds to more than oneGPCR, the test compound binds to each GPCR with a similar potency.Typically, the K_(d) values for the particular binding partner bindingto each of the respective GPCRs are within 5-10 fold of each other, suchas within 2-3 fold.

It will be appreciated that the methods of the invention allow forcombinations of test compounds that bind to one or more GPCRs to beisolated, either by repeating the method with single test compounds,providing multiple test compounds in one cycle of the method or by usinga library of test compounds in the method.

In a further embodiment, it may be advantageous to select those testcompounds which, while still able to bind to a first GPCR, are not ableto bind, or bind less strongly than to the first GPCR, to at least oneother GPCR, for example a second GPCR. It will be appreciated that thefirst and second GPCRs may be any pair of GPCRs. Thus, for example, thetest compound may be one that is selected on the basis that it binds toa first GPCR, but the test compound so selected is further tested todetermine whether it binds a second GPCR (or binds less strongly to asecond GPCR than the first GPCR). Test compounds are selected which donot bind (or have reduced binding to) the second GPCR. Where the bindingpartner is therapeutic molecule, such an approach may help to reduce thebinding partner's toxicity.

It is preferred that the test compound binds the further (second) GPCRwith an affinity which is less than 50% of the affinity the compound hasfor first GPCR, more preferably less than 10% and still more preferablyless than 1% or 0.1% or 0.01% of the affinity the compound has for thefirst GPCR. Thus, the K_(d) for the interaction of the test compoundwith the first GPCR is higher than for the second GPCR.

A second aspect of the invention provides a method for producing abinding partner of a GPCR, the method comprising synthesising a bindingpartner identifiable by carrying out the method according to the firstaspect of the invention.

The binding partners can be synthesised by any suitable method known inthe art including the techniques of organic chemistry, molecular biologyor biochemistry. For example, if the binding partner is a polypeptide,the binding partner may be made by expressing the nucleic acid moleculeencoding the binding partner in a suitable host cell as is known in theart. Antibodies may be synthesised using any of the methods describedabove including, for example, recombinant DNA technology.

A third aspect of the invention provides a binding partner obtained byany of the methods of the first aspect of the invention.

In an embodiment, the binding partner is a conformation-specific bindingpartner, as described above.

A fourth aspect of the invention provides a binding partner, for examplea conformation-specific binding partner, obtainable by any of themethods of the first aspect of the invention.

The binding partner may be any of a polypeptide; an anticalin; apeptide; an antibody; a chimeric antibody; a single chain antibody; anaptamer; a darpin; a Fab, F(ab′)₂, Fv, ScFv or dAb antibody fragment; asmall molecule; a natural product; an affibody; a peptidomimetic; anucleic acid; a peptide nucleic acid molecule; a lipid; a carbohydrate;a protein based on a modular framework including ankyrin repeatproteins, armadillo repeat proteins, leucine rich proteins,tetrariopeptide repeat proteins or Designed Ankyrin Repeat Proteins(DARPins); or proteins based on lipocalin or fibronectin domains orAffilin scaffolds based on either human gamma crystalline or humanubiquitin.

In a preferred embodiment, the binding partner is an antibody. Forexample, the antibody may be specific to a non-contiguous epitope in theGPCR or may be specific to a contiguous epitope in the GPCR. Preferably,the relevant epitopes in the parent and mutant GPCR recognised by theantibody are similar, whether they be contiguous or non-contiguous.Specifically, extracellular epitopes such as C- or N-terminii orpolypeptide loops of the parent and mutant GPCR are preferably similar.

Typically the binding partner to the mutant GPCR with a similar potencyto its binding to the parent GPCR. Typically, the K_(d) values for theparticular binding partner binding the mutant GPCR and the parent GPCRare within 5-10 fold of each other, such as within 2-3 fold. Typically,the binding of the binding partner to the mutant GPCR compared to theparent GPCR would be not more than 5 times weaker and not more than 10times stronger.

Typically, mutant receptors which have been stabilised in the selectedconformation should bind the binding partner with approximately equalaffinity (that is to say typically within 2-3 fold) or greater affinitythan does the parent receptor. For agonist-conformation bindingpartners, the mutants typically bind the agonists with the same orhigher affinity than the parent GPCR and typically bind antagonists withthe same or lower affinity than the parent GPCR. Similarly forantagonist-conformation binding partners, the mutants typically bind theantagonists with the same or higher affinity than the parent GPCR andtypically bind agonists with the same or lower affinity than the parentGPCR.

It is appreciated that the methods of the invention may be used as abiosensor to detect target substances such as molecules, especiallybiomolecules. For example, the biosensor may be used to detectbiomarkers of disease or drug treatment which may be used as adiagnostic or prognostic. The mutant GPCR may be immobilised on a sensorsurface and binding of compounds detected, for example, by surfaceplasmon resonance as described above. In a further example of abiosensor, compound binding to the receptor can be detected by changesin intrinsic tryptophan fluorescence or using fluorescence resonanceenergy transfer between an intrinsic tryptophan resident donor and afluorescent acceptor (Lakowicz J R 1999. Principles of fluorescencespectroscopy, Plenum New York, Martin D D, Budamagunta M S, Ryan R O,Voss J C, Oda M N. J Biol Chem. 2006 Jul. 21; 281(29):20418-26).Alternatively, mutant GPCRs may be used in acoustic biosensors, whereinthe mutant GPCRs are immobilised on quartz crystal resonator sensors(QCRS) and the acoustic sensor response used to detect compound—mutantGPCR binding interactions (Cooper M A, Drug Discov Today. 2006 December;11(23-24):1068-74. Epub 2006 Oct. 20).

Accordingly, a fifth aspect of the invention provides a biosensorcomprising a mutant GPCR of a parent GPCR wherein the mutant GPCR hasincreased stability in a particular conformation relative to the parentGPCR, and wherein when a target substance binds to said mutant GPCR, adetectable signal is produced.

Preferences for the mutant GPCRs and their methods of production are asdefined above with respect to the first aspect of the invention.

Preferably, the biosensor is in a chip form or a bead supported form,where the mutant GPCRs are immobilised on a chip or bead and used todetect target substances. However, it will be appreciated that themutant GPCRs may be provided in soluble form, in which case thebiosensor would comprise a solution.

Immobilisation of mutant GPCRs onto a solid support for incorporationinto a biosensor can be performed using methods well known in the artand as described above with respect to the first aspect of theinvention. Typically, mutant GPCRs are reconstituted onto chip surfacessuitable for direct biosensor analysis via flow-mediated surfacereconstitution (Karlsson et al., Analytical Biochemistry 300, 132-138(2002)). For example, rapid immobilization and reconstitution of GPCRson carboxylated dextran surfaces modified with long alkyl groups can beachieved following amine coupling of a detergent-solubilized receptor;lipid/detergent-mixed micelles are adhered as they are injected over theimmobilized surface, taking advantage of integrated flow cells presentin many biosensor systems. The detergent can then be eluted in thesubsequent buffer flow leaving functional, intact mutant GPCRs forsubsequent screening and analysis. Such mutant GPCR preparationscontaining detergent are ideally suited for use in a flow-basedbiosensor, such as quartz crystal microbalance biosensor, an evanescentwave biosensor, a planar wave guide biosensor, a surface Raman sensor,or a surface plasmon resonance biosensor. In the latter case,solubilized receptors can be captured on a GE Healthcare (Biacore) CM4or CM5 dextran sensor chip. The dextran matrix of the sensor chip isactivated by 35 uL of 50 mM N-hydroxysuccinimide and 200 mMN-ethyl-N-[(dimethylamino)propyl]carbodiimide at a flow rate of 5uL/min, followed by a 7-min injection of 0.1 mg/mL detergent-solubilisedGPCR receptor (for example with 25 mM CHAPS in 10 mM MOPS, pH 7.5). Anyremaining reactive carboxy groups are deactivated using a 7-min pulse of1 M ethanolamine hydrochloride, pH 8.5. After the injection, thebiosensor chip is washed at high flow rate with the surface plasmonresonance running solution until a stable baseline is restored (ca. 30min). This washing step works like a flow dialysis procedure and ensuresthe removal of the detergent from the sensor chip surface; however, itwill be appreciated that hydrophobic parts of the GPCR may still beattached to some lipid or detergent molecules in order to maintainfunctional integrity.

The target substance may be any of a molecule, a biomolecule, a peptide,a protein, a carbohydrate, a lipid, a GPCR ligand, a synthetic molecule,a drug, a drug metabolite or a disease biomarker.

In an embodiment, the detectable signal is any of a change in colour;fluorescence; evanescence; surface plasmon resonance; electricalconductance or charge separation; ultraviolet, visible or infraredabsorption; luminescence; chemiluminescence; electrochemiluminescence;fluorescence anisotropy; fluorescence intensity; fluorescence lifetime;fluorescence polarisation; fluorescence energy transfer; molecular mass;electron spin resonance; nuclear magnetic resonance; hydrodynamic volumeor radius; specific gravity; scintillation; field effect resistance;electrical impedance; acoustic impedance; quantum evanescence; resonantscattering; fluorescent quenching; fluorescence correlationspectroscopy; acoustic load; acoustic shear wave velocity; bindingforce; or interfacial stress.

The invention will now be described in more detail with respect to thefollowing Figures and Examples wherein:

FIG. 1 Amino acid changes in βAR that lead to thermostability. Stabilityquotient indicates the % remaining binding activity of the mutants afterheating the sample for 30 min at 32° C. All values are normalized toβAR₃₄₋₄₂₄ (50%, showed as a discontinuous line) to remove anyexperimental variability between assays. Bars show the stability foreach mutant. The letters on the x-axis indicate the amino acid presentin the mutant. The original amino acid and its position in βAR₃₄₋₄₂₄ isindicated below. Bars corresponding to the same amino acid in βAR₃₄₋₄₂₄are in the same colour with arrows indicating the best mutations. Errorswere calculated from duplicate measurements; the best mutants weresubsequently re-assayed to determine the Tm for each individual mutationand to give an accurate rank order of stability for each mutant (seeExample 1).

FIG. 2 Side chains in rhodopsin that are at equivalent positions to thethermostable mutations in βAR₃₄₋₄₂₄. The equivalent amino acid residuesin rhodopsin to the amino acid residues mutated in βAR₃₄₋₄₂₄ werelocated in the rhodopsin structure, based upon an alignment amongrhodopsin, β1 adrenergic receptor, neurotensin receptor, and adenosineA_(2a) receptor (data not shown). Side chains in the same transmembranehelix are shown as space filling models in the same colour. The name andposition of the amino acid residues are those in rhodopsin.

FIG. 3 Evolution of thermostability in βAR. Starting from βAR-m10-8,combinations of mutations were rearranged systematically to find theoptimum combination of mutations (see also Table 2).

FIG. 4 Stability of βAR-m23 and βAR₃₄₋₄₂₄ in the apo-state or containingthe bound antagonist [³H]-DHA. To determine Tm in the absence of ligand(apo-state, discontinuous lines), detergent-solubilised receptors wereincubated for 30 minutes at the temperatures indicated before carryingout the binding assay. For the Tm determination of the antagonist-boundform (continuous lines), detergent-solubilised receptors werepre-incubated with [³H]-DHA, followed by incubation at the temperaturesindicated. βAR-m23 (circles), and βAR₃₄₋₄₂₄ (squares). Data points arefrom duplicates measurements in a representative experiment.

FIG. 5 Competition binding of agonists to βAR-m23 and βAR₃₄₋₄₂₄. Bindingassays were performed on receptors partially purified in DDM; βAR-m23(triangles) and βAR₃₄₋₄₂₄ (squares). [³H]-DHA was used at aconcentration three times greater than the K_(D) of partially purifiedreceptor (see Methods). [³H]-DHA binding was competed with increasingconcentrations of the agonists, norepinephrine (a) and isoprenaline (b),or with an antagonist, alprenolol (c). Log EC₅₀ and corresponding EC₅₀values for the different ligands were calculated by nonlinear regressionusing GraphPad Prism software and the error for log EC₅₀s were lowerthan 10%. The EC₅₀s for ligand binding to βAR₃₄₋₄₂₄ and βAR-m23 are:norepinephrine, βAR₃₄₋₄₂₄ 1.5 μM, βAR-m23 3.7 mM; isoprenaline,βAR₃₄₋₄₂₄ 330 nM, βAR-m23 20 μM; alprenolol, βAR 78 nM, βAR-m23 112 nM.

FIG. 6 Stability of βAR-m23 and βAR₃₄₋₄₂₄ in five different detergents.Samples of βAR₃₄₋₄₂₄ (a), and βAR-m23 (b) solubilized in DDM werepartially purified on Ni-NTA agarose columns allowing the exchange intovarious different detergents: DDM (squares), DM (triangles), OG(inverted triangles), LDAO (diamonds) and NG (circles). βAR is sounstable in OG, NG and LDAO that it was not possible to measure anyactivity after purification at 6° C. Assays were carried out asdescribed in the Methods and the Tm is shown at the intersection betweenthe curves and the discontinuous line. Results are from duplicatemeasurements in a representative experiment performed in parallel. (c)Photomicrograph of a crystal of βAR-m23 mutant, which showed good orderby X-ray diffraction.

FIG. 7 Curve of thermostability of βAR₃₄₋₄₂₄ (Tm). Binding assays wereperformed using [³H]-dihydroalprenolol (DHA) as radioligand as describedunder “Methods”. Samples were heated for 30 minutes at differenttemperatures before the assay. Tm represents the temperature at whichthe binding decreased to the 50%, value showed as a discontinuous line.Data points are from duplicates of one single experiment. Thisexperiment has been repeated several times with similar results.

FIG. 8 Saturation binding assays of membranes of βAR₃₄₋₄₂₄ and βAR-m23.Binding assays were performed as described in “Methods” using[³H]-dihydroalprenolol (DHA) as radioligand; βAR₃₄₋₄₂₄ (a) and βAR-m23(b). Scatchard plots are shown as insets along with the correspondingvalues for B_(max) and K_(D). Data points are from duplicates of twoindependent experiments for each protein. Data were analyzed bynonlinear regression using Prism software (GraphPad).

FIG. 9 Alignment of the turkey β-adrenergic receptor with human β1, β2and β3 receptors.

FIG. 10 Alignment of human adenosine receptors.

FIG. 11 Alignment of neurotensin receptors.

FIG. 12 Flow chart showing the two different assay formats of ligand (+)and ligand (−) used to determine receptor thermostablity.

FIG. 13 Pharmacological profile of thermostable mutant adenosine A2areceptor, Rant21. Saturation binding of (A) antagonist and (B) agonistto solubilised receptors. (C-F) Inhibition of [³H]ZM241385 binding byincreasing concentrations of antagonists (C) XAC and (D) Theophylline,and agonists (E) NECA and (F) R-PIA; binding of [³H]ZM241385 (10 nM) inthe absence of unlabelled ligand was set to 100%. Each solubilisedreceptor was incubated with ligands for one hour on ice in bindingbuffer (50 mM Tris pH7.5 and 0.025% DDM) containing 400 mM NaCl (A,C-F). Data shown are from two independent experiments with each datapoint measured in triplicate. K_(D) and K_(i) values are given in Table(iii).

FIG. 14 Thermostable mutants show a decreased dependence on lipids (A)and an increased survival at higher concentration of DDM (B) uponheating compared to the wild-type receptor. Receptors were solubilisedin 1% DDM (diluted in 50 mM Tris pH7.5 and 400 mM NaCl) and immobilisedon Ni-NTA agarose for the IMAC step. Exchange of buffer containing theappropriate concentration of DDM and/or lipids was performed duringwashes and elution from the Ni-NTA beads.

FIG. 15 Mapping of the M90V, Y227A, A282L and F338M m23 mutations inturkey beta1 adrenergic receptor onto homologous residues (M82, Y219,C265 and A321 respectively) in the human beta2 adrenergic receptorstructure (Rasmussen et al (2007) Nature 15; 383-387; pdb accessioncodes 2R4R and 2R4S) reveals their position at a helical interface andhelical kink respectively. Amino acid residues in equivalent positionsto the thermostabilising mutations in the turkey β1 adrenergic receptorare shown as labelled space filling models.

FIG. 16 Mapping of m23 mutations in turkey beta1 adrenergic receptoronto homologous residues in the human beta2 adrenergic receptorstructure (Cherezov et al (2007) Science, 318:1258-65; pdb accessioncode 2RH1). The Cα trace of the β2AR is shown with the fusion moiety (T4lysozyme) removed. The six mutations in βAR-m23 (R68S, M90V, Y227A,A282L, F327A, F338M) are equivalent to amino acid residues K60, M82,Y219, C265, L310, F321 in the human β2AR. Lys60 is on the intracellularend of Helix 1 and points into the lipid-water interface. Met82 is nearthe middle of Helix 2 and points into the ligand binding pocket; thenearest distance between the substrate carazolol and the Met side chainis 5.7 Å. Tyr219 is towards the intracellular end of helix 5 and is atthe helix5-helix 6 interface. Cys265 is at the end of the loop regionbetween helices 5 and 6 and points away from the transmembrane regions.Leu310 and Phe321 are both in helix 7 and both point out into the lipidbilayer.

FIG. 17 Multiple sequence alignment of human beta-2AR, rat NTR1, turkeybeta-1 AR, human Adenosine A2aR and human muscarinic M1 receptors. Ineach sequence, thermostabilising mutations are marked with a box.Mutations occurring in two or more sequences are denoted with a star.

FIG. 18 Mapping of turkey beta1AR mutation I55A (human beta2AR I47) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is at theinterface between 3 helices (H1, H2 kink, H7 kink). Left: side view;right: top view.

FIG. 19 Mapping of turkey beta1AR V89L mutation (human beta2AR V81) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is in thekink in helix 2. The helices are numbered and the bound antagonist isshown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left: side view; right: top view.

FIG. 20 Mapping of turkey beta1AR M90V mutation (human beta2AR M82) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is in kinkin helix 2 oriented towards the binding pocket. The helices are numberedand the bound antagonist is shown as a space filling model. Amino acidresidues in equivalent positions to the thermostabilising mutations inthe turkey β1 adrenergic receptor are shown as space filling models andare arrowed for clarity. Left: side view; right: top view.

FIG. 21 Mapping of turkey beta1AR I129V mutation (human beta2AR I121)onto human beta2AR structure (pdb accession code 2RH1). Mutation isopposite a kink in helix 5. The helices are numbered and the boundantagonist is shown as a space filling model. Amino acid residues inequivalent positions to the thermostabilising mutations in the turkey β1adrenergic receptor are shown as space filling models and are arrowedfor clarity. Left: side view; right: bottom view.

FIG. 22 Mapping of turkey beta1AR F338M mutation (human beta2AR F321)onto human beta2AR structure (pdb accession code 2RH1). Mutation is inkink in helix 7. The helices are numbered and the bound antagonist isshown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left: side view; right: top view.

FIG. 23 Mapping of turkey beta1AR Y227A mutation (human beta2AR Y219)onto human beta2AR structure (pdb accession code 2RH1). Mutation is athelix-helix interface. The helices are numbered and the bound antagonistis shown as a space filling model. Amino acid residues in equivalentpositions to the thermostabilising mutations in the turkey β1 adrenergicreceptor are shown as space filling models and are arrowed for clarity.Left side view; right: bottom view.

FIG. 24 Mapping of turkey beta1AR A282L mutation (human beta2AR C265)onto human beta2AR structure (pdb accession code 2RH1). Mutation is inloop region. The helices are numbered and the bound antagonist is shownas a space filling model. Amino acid residues in equivalent positions tothe thermostabilising mutations in the turkey β1 adrenergic receptor areshown as space filling models and are arrowed for clarity. Left: sideview; right: top view.

FIG. 25 Mapping of turkey beta1AR R68S mutation (human beta2AR K60) ontohuman beta2AR structure (pdb accession code 2RH1). Mutation is at thelipid-water boundary, pointing into the solvent. The helices arenumbered and the bound antagonist is shown as a space filling model.Amino acid residues in equivalent positions to the thermostabilisingmutations in the turkey β1 adrenergic receptor are shown as spacefilling models and are arrowed for clarity. Left side view; right:angled top view.

FIG. 26 Comparison of the thermostabilities of three β adrenergicreceptors (turkey β1 (▪), human β1 (▾) and human β2 (•) and twothermostabilised receptors (turkey β1-m23 (▴) and human β2-m23 (♦). Thesix thermostabilising mutations in β1-m23 (R68S, M90V, Y227A, A282L,F327A, F338M) were all transferred directly to the human β2 receptor(K60S, M82V, Y219A, C265L, L310A, F321M) making β2-m23, based upon thealignment in FIG. 9. The resulting mutants were transiently expressed inmammalian cells, solubilised in 0.1% dodecylmaltoside and assayed forthermostability in the minus-ligand format (heating the apo-state,quenching on ice, adding 3H-DHA). The apparent Tms for turkey β1 andβ2-m23 were 23° C. and 45° C. respectively, giving a ΔTm of 22° C. asseen previously in E. coli expressed receptor. The Tms for human β2 andβ2-m23 were 29° C. and 41° C. respectively, showing that the aporeceptor was stabilised by 12° C. This exemplifies the principle of thetransferability of thermostabilising mutations from one receptor toanother receptor, which in this case are 59% identical. The human β1receptor (Tm˜12° C.) is much less stable than the turkey β1 receptor.

FIG. 27 Percentage identity of the turkey β1 adrenergic receptor, humanadenosine receptor and rat neurotensin receptor to human β adrenergicreceptors, human adenosine receptors and human neurotensin receptors,respectively.

FIG. 28 Alignment of neurotensin receptors

FIG. 29 Schematic representation of typical lead isolation process forthe identification of inhibitory scFv binders.

FIG. 30 Results of (A) polyclonal and (B) monoclonal phage ELISAs. (B)Columns 1-9 rows A-H of a 96 well ELISA plate were used to screen 69anti β-AR phage clones

FIG. 31 Specificity phage ELISA using β-andregenic receptor and 3unrelated control proteins (CD86-CD4, Notch1-Fc and the NRR region ofNotch1). Proteins coated on two ELISA plates, amino-plate (A) andHis-plate (B) are shown. Beta-AR phage clones (hashed bars) from left toright (C1, E2, A3, G3, C4, D4, F4, H4, D5, F5, G5, C6, D6, C7, F7, B8and C8) names originates from the screen shown in FIG. 30. In the graph,sticky anti β-AR clones are indicated by their clone names over thecorresponding bar. Also shown are binding of control phage populationsspecific to the control proteins, anti-CD86 (grey bars), anti-N1 EGF(white bars) and anti-N1 NRR (dotted bars).

FIG. 32 Anti-β-AR antibody clones (white bars) and positive controlantibody (black bar) and a no antibody negative control (black bar) areshown.

FIG. 33 Two capture stages of the biotinylated β1AR onto astreptavidin-coated flow cell. A ˜1200 RU captured; B ˜4000 RU captured.

FIG. 34 Biacore responses for alprenolol. The highest concentration is666 nM and each concentration was tested three times in a three-folddilution series. The responses are concentration dependent and fairlyreproducible. The lines depict the fit of a simple 1:1 interaction modeland the parameters determined from this fit are listed in the inset (thenumber in parentheses is the error in the last digit).

FIG. 35 Normalised responses to alprenolol. The responses are normalizedwith respect to the Rmax determined (from the fitting) for each curve.

FIG. 36 Biacore responses for propranolol. The highest concentration is111 nM and each concentration was tested two or three times in athree-fold dilution series.

FIG. 37 Normalised responses to propranol. The responses are normalizedwith respect to the Rmax determined (from the fitting) for each curve.

FIG. 38 Biacore responses to alprenolol on β1AR surface that was almostthree days old.

FIG. 39 Capture of β1AR to a density of 8000 RU.

FIG. 40 Biacore response to alprenolol binding to 8000 RU β1AR surface.

FIG. 41 Biacore responses to salmeterol using 2 different surfaces.Salmeterol was tested using a highest concentration of 1.67 uM, witheach concentration tested two or three times. A. 4200 RU surface; B.8000 RU surface. Larger responses are observed with the 8000 RU surface.

FIG. 42 Biacore responses for salmeterol, shown as normalised responses.A. 4200 RU surface; B. 8000 RU surface.

FIG. 43 Biacore responses to isoproterenol. Isoproterenol was testedusing a highest concentration of 2 μM. A. 4200 RU surface. B. 8000 RUsurface.

FIG. 44 Biacore responses for carvedilol. A. 5200 RU surface; B. 8000 RUsurface.

FIG. 45 Competition binding curves to a range of compounds tested foractivity at the stabilised adenosine A2a receptor (Rant22) using[³H]ZM241385.

EXAMPLE 1 Conformational Stabilisation of the β-Adrenergic Receptor inDetergent-Resistant Form Summary

There are over 500 non-odorant G protein-coupled receptors (GPCRs)encoded by the human genome, many of which are predicted to be potentialtherapeutic targets, but there is only one structure available, that ofbovine rhodopsin, to represent the whole of the family. There are manyreasons for the lack of progress in GPCR structure determination, but wehypothesise that improving the detergent-stability of these receptorsand simultaneously locking them into one preferred conformation willgreatly improve the chances of crystallisation. A generic strategy forthe isolation of detergent-solubilised thermostable mutants of a GPCR,the β-adrenergic receptor, was developed based upon alanine scanningmutagenesis followed by an assay for receptor stability. Out of 318mutants tested, 15 showed a measurable increase in stability. Afteroptimisation of the amino acid residue at the site of each initialmutation, an optimally stable receptor was constructed by combiningspecific mutations. The most stable mutant receptor, βAR-m23, contained6 point mutations that led to a Tm 21° C. higher than the native proteinand, in the presence of bound antagonist, βARm23 was as stable as bovinerhodopsin. In addition, βAR-m23 was significantly more stable in a widerange of detergents ideal for crystallisation and was preferentially inan antagonist conformation in the absence of ligand.

Results

Selection of Single Mutations that Increase the Thermostability of theβ1 Adrenergic Receptor

βAR from turkey erythrocytes is an ideal target for structural studiesbecause it is well characterised and is expressed at high-levels ininsect cells using the baculovirus expression system[10,11]. The bestoverexpression of βAR is obtained using a truncated version of thereceptor containing residues 34-424 (βAR₃₄₋₄₂₄) [9] and this was used asthe starting point for this work. Alanine scanning mutagenesis was usedto define amino residues in βAR₃₄₋₄₂₄ that, when mutated, altered thethermostability of the receptor; if an alanine was present in thesequence it was mutated to a leucine residue. A total of 318 mutationswere made to amino acid residues 37-369, a region that encompasses allseven transmembrane domains and 23 amino acid residues at the Cterminus; mutations at 15 amino residues were not obtained due to strongsecondary structure in the DNA template. After sequencing each mutant toensure the presence of only the desired mutation, the receptors werefunctionally expressed in E. coli and assayed for stability.

The assay for thermostability was performed on unpurifieddetergent-solubilised receptors by heating the receptors at 32° C. for30 minutes, quenching the reaction on ice and then performing aradioligand binding assay, using the antagonist [³H]-dihydroalprenolol,to determine the number of remaining functional βAR₃₄₋₄₂₄ moleculescompared to the unheated control. Heating the unmutated βAR₃₄₋₄₂₄ at 32°C. for 30 min before the assay reduced binding to approximately 50% ofthe unheated control (FIG. 7); all the data for the mutants werenormalised by including the unmutated βAR₃₄₋₄₂₄ as a control in everyassay performed. In the first round of screening, eighteen mutantsshowed an apparent increase in stability, maintaining more than 75% ofantagonist binding after heating and being expressed in E. coli to atleast 50% of the native βAR₃₄₋₄₂₄ levels. In view of the possibility ofincreasing further the stability of these mutants, each of the 18residues was mutated to 2-5 alternative amino acid residues of varyingsize or charge (FIG. 1). Out of these 18 mutants, 12 were not improvedby further changes, 5 had better thermostability if another amino acidwas present and one mutation from the first screen turned out to be afalse positive. In addition, three residues that were not stabilisedupon mutation to alanine (V89, S151, L221) were mutated to a range ofother amino acid residues; the two positions that when mutated toalanine did not affect thermostability, were also unaffected by otherchanges. In contrast, V89 showed less thermostability when mutated toalanine, but thermostability increased when it was mutated to Leu. Thusthe initial alanine scanning successfully gave two-thirds of the bestamino acid residues of those tested for any given position.

The position and environment predicted for each of the 16 amino residuesthat gave the best increases in thermostability when mutated weredetermined by aligning the βAR sequence with that of rhodopsin whosestructure is known (FIG. 2). Fourteen of these residues were predictedto be present in transmembrane α-helices, with five of the residuespredicted to be lipid-facing, 4 being deeply buried and the remainderwere predicted to be at the interfaces between the helices. Some ofthese residues would be expected to interact with each other in the βARstructure, such as the consecutive amino acids G67 and R68 (V63 and Q64in rhodopsin), or the amino acids within the cluster Y227, R229, V230and A234 in helix 5 (Y223, Q225, L226 and V230 in rhodopsin). Otheramino acid residues that could interact in βAR were Q194A in externalloop 2 and D322A in external loop 3 (G182 and P285 in rhodopsin,respectively).

The increase in stability that each individual mutation gave toβAR₃₄₋₄₂₄ was determined by measuring the Tm for each mutant (resultsnot shown); Tm in this context is the temperature that gave a 50%decrease in functional binding after heating the receptor for 30minutes. Each mutation increased the Tm of βAR₃₄₋₄₂₄ by 1-3° C., withthe exception of M90A and Y227A that increased the Tm by 8° C.

Combining Mutations to Make an Optimally Stable Receptor

Initially, mutations that improved thermostability that were adjacent toone another in the primary amino sequence of βAR were combined.Constructions containing the mutations G67A and R68S, or differentcombinations of the mutations at the end of helix 5 (Y227A, R229Q, V230Aand A234L) were expressed and assayed; the Tm values (results not shown)were only 1-3° C. higher than the Tm for βAR₃₄₋₄₂₄ and one mutant wasactually slightly less stable, suggesting that combining mutations thatare adjacent to one another in the primary amino acid sequence does notgreatly improve thermostability. Subsequently, mutations predicted to bedistant from one another in the structure were combined. PCR reactionswere performed using various mixes of primers to combine up to 5different mutations in a random manner and then tested forthermostability (Table 1). The best of these combinations increased theTm more than 10° C. compared to the Tm of βAR₃₄₋₄₂₄. In some cases,there was a clear additive effect upon the Tm with the sequentialincorporation of individual mutations. This is seen in a series of 3mutants, m4-1, m4-7 and m4-2, with the addition of V230A to m4-1increasing the Tm by 2° C. and the additional mutation D332A in m4-7increasing the Tm a further 3° C. Mutants that contained Y227A and M90Aall showed an increase in Tm of 10° C. or more. Just these two mutationstogether increased the Tm of βAR₃₄₋₄₂₄ by 13° C. (m7-5), however, thetotal antagonist binding was less than 50% of βAR₃₄₋₄₂₄ suggestingimpaired expression of this mutant. The addition of F338M to m7-5 didnot increase the thermostability, but it increased levels of functionalexpression in E. coli.

TABLE 1 Combinations of mutations by PCR. 10 PCR reactions wereperformed combining different pairs of primers that contained theselected mutations. Successful PCR reactions are shown in the table. Thestability of these new mutants was assayed as described in FIG. 7 andthe Tm calculated. The results are shown as the mean ± S.E. fromduplicates. PCR Receptor Mutations T_(m) (° C.) βAR₃₄₋₄₂₄ 31.7 ± 0.1 4m4-1 G67A, G98A 35.5 ± 0.9 m4-2 G67A, G98A, V230A, D322A 40.9 ± 0.9 m4-6G98A, D322A 35.0 ± 0.2 m4-7 G67A, G98A, V230A 38.0 ± 1.2 6 m6-1 Y227A,A234L, A282L, A334L 41.6 ± 0.9 m6-4 R68S, Y227A, A234L, A282L 41.6 ± 0.1m6-5 R68S, A234L, A282L, A334L 41.9 ± 0.5 m6-9 R68S, Y227A, A234L,A282L, A334L 43.7 ± 0.4 m6-10 R68S, Y227A, A282L, A334L 47.4 ± 1.1 m6-11R68S, A282L, A334L 39.1 ± 0.5 7 m7-1 M90V, A282L, F338M 43.0 ± 0.8 m7-2M90V, A282L 38.9 ± 0.6 m7-5 M90V, Y227A 45.2 ± 1.0 m7-6 M90V, I129V 40.0± 0.6 m7-7 M90V, Y227A, F338M 45.2 ± 2.0 10 m10-4 R68S, M90V, V230A,A334L 46.9 ± 1.0 m10-8 R68S, M90V, V230A, F327A, A334L 47.3 ± 1.4

The most thermostable mutants obtained, which were still expressed athigh levels in E. coli, were m6-10, m7-7 and m10-8. These mutantscontained collectively a total of 10 different mutations, with 8mutations occurring in at least two of the mutants. A second round ofmutagenesis was performed using m10-8 as the template and adding orreplacing mutations present in m6-10 and m7-7 (FIG. 3); some of thesemutations were very close in the primary amino acid sequence of βAR andtherefore were not additive as noted above, but many mutations improvedthe Tm further (Table 2). For example, exchanging two mutations inm10-8, to create m18, raised the Tm to 49.6° C. and adding A282L to makem23 increased the Tm a further 3° C. to 52.8° C. This produced the mostthermostable βAR₃₄₋₄₂₄ mutant so far and will be referred to as βAR-m23.

TABLE 2 Improvement of best combination of mutations. These new mutantswere obtained mixing the changes present in mutants m6-10, m7-7 andm10-8 by PCR. The stability of these new mutants was assayed asdescribed in FIG. 7 and the Tm calculated. The results are shown as themean ± S.E. from duplicates. Mutations T_(m) (° C.) m17 R68S M90V Y227AV230A — F327A A334L — 48.2 ± 1.4 m18 R68S M90V Y227A V230A A282L F327A —F338M 49.6 ± 0/9 m19 R68S M90V Y227A — A282L F327A — F338M 49.0 ± 0.8m20 R68S M90V — — — F327A A334L — 48.4 ± 0.7 m21 R68S M90V Y227A — —F327A A334L — 47.0 ± 1.3 m22 R68S M90V Y227A F327A A334L — 47.4 ± 0.5m23 R68S M90V Y227A — A282L F327A — F338M 52.8 ± 1.4

The thermostability assays used to develop βAR₃₄₋₄₂₄ mutants wereperformed by heating the receptor in the absence of the antagonist, butit is well known that bound ligand stabilises receptors. Therefore,stability assays for βAR₃₄₋₄₂₄ and βAR-m23 were repeated with antagonistbound to the receptors during the heating step (FIG. 4). As expected,the Tm of the receptor that contained bound antagonist during theincubation was higher than that for the receptor without antagonist. ForβAR₃₄₋₄₂₄ the Tm was 6° C. higher with bound antagonist and for βAR-m23the Tm increased 2° C. to 55° C.; the smaller increase inthermostability observed for βAR-m23 when antagonist binds suggests thatthe receptor is already in a more stable conformation similar to theantagonist bound state than βAR₃₄₋₄₂₄ (see also below). The Tm ofβAR-m23 with antagonist bound is very similar to the Tm of dark-staterhodopsin in dodecylmaltoside (DDM)[12], whose structure has been solvedby two independent laboratories[13,14]. This suggested that βAR-m23 issufficiently stable for crystallisation.

Characterization of βAR-m23

The three characteristic activities measured for βAR-m23 and βAR₃₄₋₄₂₄to identify the effect of the six mutations were the affinity ofantagonist binding, the relative efficacies of agonist binding and theability of βAR-m23 to couple to G proteins.

Saturation binding experiments to membranes using the antagonist[³H]-dihydroalprenolol (FIG. 8) showed that the affinity of binding toβAR-m23 (K_(D) 6.5±0.2 nM, n=2) was slightly lower than for βAR₃₄₋₄₂₄(K_(D) 2.8±0.1 nM, n=2), suggesting that there are no largeperturbations in the structure of βARm23 in the antagonist-boundconformation. This is consistent with the observation that none of themutations in βAR-m23 correspond with amino acids believed to beimplicated in ligand binding. In contrast to antagonist binding, theefficacy of agonist binding by βAR-m23 is 3 orders of magnitude weakerthan for βAR₃₄₋₄₂₄ (FIG. 5). The potency of the agonist isoprenaline isconsistently lower in βAR-m23 and βAR₃₄₋₄₂₄ than for the native agonistnorepinephrine, indicating that the agonist-bound conformation for thetwo receptors is likely to be similar. However, the large decrease inagonist efficacy in βAR-m23 compared to βAR₃₄₋₄₂₄ indicates that the 6mutations in βAR-m23 have locked the receptor preferentially in anantagonist-bound conformation. From a crystallisation perspective, thisis an added bonus to thermostabilisation, because it is essential tohave a conformationally homogeneous protein population for theproduction of diffraction-quality crystals.

All of the thermostability assays used to derive βAR-m23 were performedon receptors solubilised in DDM. The aim of the thermostabilisationprocess was to produce a receptor that is ideal for crystallography,which means being stable in a variety of different detergents and notjust DDM. We therefore tested the stability of βAR-m23 and βAR in avariety of different detergents, concentrating on small detergents thatare preferentially used in crystallising integral membrane proteins.Membranes prepared from E. coli expressing βAR-m23 or βAR₃₄₋₄₂₄ weresolubilised in DDM, bound to Ni-NTA agarose then washed with either DDM,decylmaltoside (DM), octyiglucoside (OG), lauryldimethylamine oxide(LDAO) or nonylglucoside (NG). Stability assays were performed on thereceptors in each of the different detergents (FIG. 6). βAR₃₄₋₄₂₄ wasonly stable in DDM and DM, with no active receptors eluting from theresin washed with OG, NG or LDAO. In contrast, functional βAR-m23 wasstill present in all detergents and the Tm could be determined. Asexpected, the smaller detergents were considerably more denaturing thaneither DDM (Tm 52° C.) or DM (Tm 48° C.), with T_(m)s of 25° C. (NG),23° C. (LDAO) and 17° C. (OG). The difference in Tm between βAR-m23 andβAR₃₄₋₄₂₄ is about 20° C., irrespective of whether the receptors weresolubilised in either DDM or DM; it is therefore not surprising that noactive βAR₃₄₋₄₂₄ could be found in even NG, because the predicted Tmwould be about 5° C., thus resulting in rapid inactivation of thereceptor under the conditions used for purification. The selectionstrategy used for the generation of βAR-m23 was chosen deliberately tobe based upon thermostability, because it is far simpler to apply thanselecting for stability in detergents of increasing harshness. However,it is clear that increasing the thermostability of βAR₃₄₋₄₂₄ alsoresulted in increasing tolerance to small detergents ideal forcrystallising integral membrane proteins.

Crystallisation of Mutant GPCR

Earlier attempts to crystallise several different constructs of turkeybeta-adrenegic receptor failed. Despite experimenting with a variety ofconditions, using both the native sequence and several truncated andloop-deleted constructs, over many years, no crystals were obtained.

However, once the stabilising mutations from βAR-m23 were transferredinto the constructs, several different crystals were obtained indifferent detergents and different conditions.

The crystals that have been most studied so far were obtained using thepurified beta-36 construct (amino acid residues 34-367 of the turkeybeta receptor containing the following changes: point mutations C116Land C358A; the 6 thermostabilising point mutations in m23; replacementof amino acid residues 244-278 with the sequence ASKRK; a C terminalHis6 tag) expressed in insect cells using the baculovirus expressionsystem, after transferring the receptor into the detergentoctyl-thioglucoside. The precipitant used was PEG600 or PEG1000 and thecrystals obtained are elongated plates.

Experiments have also been carried out to see whether, once thecrystallisation conditions had been defined using the stabilisedreceptor, it was possible to get crystals using the originalnon-stablised construct. It was possible that similar or perhaps verysmall crystals could have been obtained, but, in fact, the “wild type”(i.e. the starting structure from which the mutagenesis began) nevergave any crystals.

The crystals are plate-shaped with space group C2 and diffract well,though the cell dimensions do vary depending on the freezing conditionsused.

In general, once a GPCR has been stabilised it may be subjected to avariety of well-known techniques for structure determination. The mostcommon technique for crystallising membrane proteins is by vapourdiffusion (20, 21), usually using initially a few thousandcrystallisation conditions set up using commercial robotic devices (22).However, sometimes the crystals formed by vapour diffusion are small anddisordered, so additional techniques may then be employed. One techniqueinvolves the co-crystallisation (by vapour diffusion) of the membraneprotein with antibodies that bind specifically to conformationalepitopes on the proteins' surface (23, 24); this increases thehydrophilic surface of the protein and can form strong crystal contacts.A second alternative is to use a different crystallisation matrix thatis commonly called either lipidic cubic phase or lipidic mesophase (25,26), which has also been developed into a robotic platform (27). Thishas proven very successful for producing high quality crystals ofproteins with only small hydrophilic surfaces e.g. bacteriorhodopsin(28). Membrane protein structures can also be determined tohigh-resolution by electron crystallography (29).

The evolution of βAR-m23 from βAR₃₄₋₄₂₄ by a combination of alaninescanning mutagenesis and the selection of thermostable mutants hasresulted in a GPCR that is ideal for crystallography. The Tm for βAR-m23is 21° C. higher than for βAR₃₄₋₄₂₄ and, in the presence of antagonist,βAR-m23 has a similar stability to rhodopsin. The increased Tm ofβAR-m23 has resulted in an increased stability in a variety of smalldetergents that inactivate βAR₃₄₋₄₂₄. In addition, the selectionstrategy employed resulted in a receptor that is preferentially in theantagonist-bound conformation, which will also improve the chances ofobtaining crystals, because the population of receptor conformationswill be more homogeneous than for wild type βAR₃₄₋₄₂₄. Thus we haveachieved a process of conformational stabilisation in a single selectionprocedure.

It is not at all clear why the particular mutations we have introducedlead to the thermostabilisation of the receptor. Equivalent positions inrhodopsin suggest that the amino acid residues mutated could be pointinginto the lipid bilayer, into the centre of the receptor or at theinterfaces between these two environments.

Given the difficulties in trying to understand the complexities of thethermostabilisation of soluble proteins[15], it seems unlikely thatmembrane proteins will be any easier to comprehend; we found that therewas no particular pattern in the amino acid residues in βAR that, whenmutated, led to thermostability. However, since nearly 5% of the mutantsproduced were more stable than the native receptor, alanine scanningmutagenesis represents an efficient strategy to rapidly identifythermostable mutants.

The procedure we have used to generate βAR-m23 is equally applicable toany membrane protein that has a convenient assay for detecting activityin the detergent solubilized form. While we have selected for stabilityas a function of temperature as the most convenient primary parameter,the procedure can easily be extended to test primarily for stability,for example, in a harsh detergent, an extreme of pH or in the presenceof chaotropic salts. Conformational stabilisation of a variety of humanreceptors, channels and transporters will make them far more amenable tocrystallography and will also allow the improvement in resolution ofmembrane proteins that have already been crystallised. It is to be hopedthat conformational stabilisation will allow membrane proteincrystallisation to become a far more tractable problem with a greaterprobability of rapid success than is currently the case. This shouldallow routine crystallisation of human membrane proteins in thepharmaceutical industry, resulting in valuable structural insights intodrug development.

Methods

Materials. The truncated β1 adrenergic receptor from turkey(βAR₃₄₋₄₂₄)[9] was kindly provided by Dr Tony Warne (MRC Laboratory ofMolecular Biology, Cambridge, UK). This βAR construct encoding residues34-424 contains the mutation C116L to improve expression[11], and aC-terminal tag of 10 histidines for purification.1-[4,6-propyl-³H]-dihydroalprenolol ([³H]-DHA) was supplied by AmershamBioscience, (+) L-norepinephrine bitartrate salt, (−) isoprenalinehydrochloride, (−) alprenolol tartrate salt and s-propranololhydrochloride were from Sigma.

Mutagenesis of βAR. The βAR cDNA was ligated into pRGIII to allow thefunctional expression of βAR in E. coli as a MalE fusion protein[16].Mutants were generated by PCR using the expression plasmid as templateusing the QuikChange II methodology (Stratagene). PCR reactions weretransformed into XL10-Gold ultracompetent cells (Stratagene) andindividual clones were fully sequenced to check that only the desiredmutation was present. Different mutations were combined randomly by PCRby including all the pairs of primers that introduced the followingmutations: Mut4, G67A, G068A, V230A, D322A and F327A; Mut6, R068S,Y227A, A234L, A282L and A334L; Mut7, M90V, I129V, Y227A, A282L andF338M; Mut10, R68S, M90V, V230A, F327A and A334L. The PCR mixes weretransformed and the clones sequenced to determine exactly whichmutations were introduced.

Protein expression and membrane preparations. Expression of βAR and themutants was performed in XL10 cells (Stratagene). Cultures of 50 ml of2xTY medium containing ampicillin (100 μg/ml) were grown at 37° C. withshaking until OD₆₀₀=3 and then induced with 0.4 mM IPTG. Inducedcultures were incubated at 25° C. for 4 h and then cells were harvestedby centrifugation at 13,000×g for 1 min (aliquots of 2 ml) and stored at−20° C. For the assays, cells were broken by freeze-thaw (five cycles),resuspended in 500 μl of buffer [20 mM Tris pH 8, 0.4 M NaCl, 1 mM EDTAand protease inhibitors (Complete™, Roche)]. After an incubation for 1 hat 4° C. with 100 μg/ml lysozyme and DNase I (Sigma), samples weresolubilized with 2% DDM on ice for 30 minutes. Insoluble material wasremoved by centrifugation (15,000×g, 2 min, 4° C.) and the supernatantwas used directly in radioligand binding assays.

For large-scale membrane preparations, 2L and 6L of E. coli culture ofβAR and Mut23, respectively, were grown as described above. Cells wereharvested by centrifugation at 5,000×g for 20 min, frozen in liquidnitrogen and stored at −80° C. Pellets were resuspended in 10 ml of 20mM Tris pH 7.5 containing 1× protease inhibitor cocktail (Complete™EDTA-free, Roche); 1 mg DNase I (Sigma) was added and the final volumewas made to 100 ml. Cells were broken by a French press (2 passages,20,000 psi), and centrifuged at 12,000×g for 45 min at 4° C. to removecell debris. The supernatant (membranes) was centrifuged at 200,000×gfor 30 min at 4° C.; the membrane pellet was resuspended in 15 ml of 20mM Tris pH 7.5 and stored in 1 ml aliquots at −80° C. afterflash-freezing in liquid nitrogen. The protein concentration wasdetermined by the amido black method[17]. These samples were used inradioligand binding assays after thawing and being solubilized in 2% DDMas above.

For competition assays, as well as testing different detergents,DDM-solubilized βAR was partially purified with Ni-NTA agarose (Qiagen).200 μl of Ni-NTA agarose was added to 2 ml of solubilized samples (10mg/ml of membrane protein) in 20 mM Tris pH 8, 0.4 M NaCl, 20 mMimidazole pH 8 and incubated for 1 h at 4° C. After incubation, sampleswere centrifuged at 13,000×g for 30 sec and washed twice with 250 μl ofbuffer (20 mM Tris pH 8, 0.4 M NaCl, 20 mM imidazole) containingdetergent (either 0.1% DDM, 0.1% DM, 0.1% LDAO, 0.3% NG or 0.7% OG).

Receptors were eluted in 2×100 μl of buffer (0.4 M NaCl, 1 mM EDTA, 250mM imidazole pH 8, plus the relevant detergent). The K_(D) for [³H]-DHAbinding to semipurified βAR₃₄₋₄₂₄ and βAR-m23 was, respectively 3.7 nMand 12.5 nM and the final concentration of [³H]-DHA used in thecompetition assays was 3 times the K_(D) ie 12 nM for βAR₃₄₋₄₂₄ and 40nM for βAR-m23.

Radioligand binding and thermostability assays. Single point bindingassays contained 20 mM Tris pH 8, 0.4 M NaCl, 1 mM EDTA, 0.1% DDM (orcorresponding detergent) with 50 nM [³H]-DHA and 20-100 μg membraneprotein in a final volume of 120 μl; equilibration was for 1 h at 4° C.Thermostability was assessed by incubating the binding assay mix, withor without [³H]-DHA at the specified temperature for 30 minutes;reactions were placed on ice and [³H]-DHA added as necessary andequilibrated for a further hour. Receptor-bound and free radioligandwere separated by gel filtration as described previously[18].Non-specific binding was determined in the presence of 1 μM ofs-propranolol. Saturation curves were obtained using a range of [³H]-DHAconcentration from 0.4 nM to 100 nM. Competition assays were performedusing a concentration of [³H]-DHA of 12 nM for βAR₃₄₋₄₂₄ and 40 nM forβAR-m23 (ie three times the K_(D)) and various concentrations ofunlabeled ligands (0-100 mM). Radioactivity was counted on a BeckmanLS6000 liquid scintillation counter and data were analyzed by nonlinearregression using Prism software (GraphPad).

Location of βAR-m23 thermostable mutations in rhodopsin structure. Thepdb file for the rhodopsin structure, accession code 1GZM[14], wasdownloaded from the Protein Data Bank website (www.pdb.org) anddisplayed in the program PyMOLX11Hybrid (DeLano Scientific). Theequivalent amino acid residues in rhodopsin for the thermostablemutations in βAR were located in the rhodopsin structure based upon analignment among the four GPCRs with which we are most familiar, namelyrhodopsin, β1 adrenergic receptor, neurotensin receptor and adenosineA_(2a) receptor[19].

EXAMPLE 2 Mutants of the Adenosine A_(2a) Receptor (A_(2a)R) withIncreased Thermostability

-   1. 315 site-directed mutants made between residues 2-316 of A_(2a)R.-   2. All of these mutants have been assayed for thermostability using    an assay measuring agonist and antagonist binding after the heating    step (Ligand(−) format as described in FIG. 12).    -   a. 26 mutants showed improved thermostability when measured with        ³H-NECA (agonist): G114 A, G118A, L167A, A184L, R199A, A203L,        L208A, Q210A, S213A, E219A, R220A, S223A, T224A, Q226A, K227A,        H230A, L241A, P260A, S263A, L267A, L272A, T279A, N284A, Q311A,        P313A, K315A.    -   b. 18 mutants showed improved thermostability when assayed with        ³H-ZM241385 (antagonist): A54L, V57A, H75A, T88A, G114A, G118A,        T119A, K122A, G123A, P149A, E151A, G152A, A203L, A204L, A231L,        L235A, V239A.-   3. Mutations have been combined to generate mutants in a putative    antagonist conformation. Wildtype A_(2a)R has a Tm of 31° C. with    ZM241385 bound.

a. Rant17 A54L + K122A + L235A Tm 48° C. (ZM241385 bound) b. Rant19A54L, T88A, V239A + Tm 47° C. (ZM241385 bound) A204L c. Rant21 A54L,T88A, V239A + Tm 49° C. (ZM241385 bound) K122A

-   4. Mutations from the agonist screen have been combined, but have    led to only a very low level of improvement in Tm of +2° C.

TABLE (i) List of A2aR stabilising mutations.

Mutants were expressed in E. coli, solubilised in 2% DDM + 10% glyceroland tested for ligand-binding, using the agonist [³H]-NECA (on theright) and the antagonist [³H]-ZM241385 (left). Concentrations ofradioligands were 6-10-fold above their K_(D) measured for the wild-typereceptor. Expression of active receptor was evaluated by ligand bindingat 4° C. Stability was assayed by heating the solubilised receptor inits apo-state at 30° C. for 30 minutes and then measuring residualbinding activity. Under these conditions, wild-type activity decays to50% (S.D. = 15%). Data obtained for expression and stability werenormalised to wild-type values. Mutations included in subsequent roundsof mutagenesis were those whose expression was ≧30-40% and stability≧130-140% compared to the wild-type. Bold lines indicate cluster ofmutations.

TABLE ii Stability of best combinations. Tm (° C.) Tm (° C.) − + − +agonist agonist antagonist antagonist Wt 21 29 wt 31 32 Rag 1 26 34 Rant5 42 46 (A184L/R199A/L272A) (A54L/T88A/V239A) Rag 23 22 38 Rant 21 41 49(Rag 1 + F79A/L208A) (Rant 5 + K122A) Receptors were solubilised in 1%DDM (no glycerol). A melting profile was obtained by heating thesolubilised receptor at different temperatures in absence (apo-state) orpresence of ligand (ligand-occupied state). Data shown arerepresentative of at least three independent experiments. S.D. is <1° C.

TABLE (iii) Summary of results for competition assays of detergent-solubilised wild-type A2aR and thermo-stable mutant Rant 21. K_(i) (M)Competitor wt Rant 21 XAC 2.3 × 10⁻⁶ 2.3 × 10⁻⁶ Theophylline 1.5 × 10⁻³0.9 × 10⁻³ NECA 7.0 × 10⁻⁶  >1 × 10⁻¹ R-PIA 1.6 × 10⁻⁵ 3.6 × 10⁻³ Valuesare representative of two independent experiments. Each data point wasassayed in triplicate and plotted as mean ± SD. Each solubilisedreceptor was incubated with ligands for one hour on ice in bindingbuffer (50 mM Tris pH 7.5 and 0.025% DDM) containing 400 mM NaCl.Binding of [3H]ZM241385 (10 nM) in the absence of unlabeled ligand wasset to 100%. Data shown are from two independent experiments with eachdata point measured in triplicate. Incubation of samples with ligandswas for 1 hour on ice with [³H]ZM241385 at a concentration of 10 nM.K_(i) values were calculated according to the Cheng and Prusoff equationusing the non-linear regression equation of the software Prism, applyinga K_(D) for [³H]ZM241385 of 12 nM for the wild-type and 15 nM for Rant21. Rant 21 did not bind NECA sufficiently for an accurate K_(i)determination (hence indicated as >1 × 10⁻¹). The affinity of Rant21 foragonist binding is weakened 232 fold for R-PIA and at least by 1900 foldfor NECA.

TABLE (iv) Summary of results for saturation assays of detergent-solubilised wild-type A2aR and thermo-stable mutants. K_(D) (nM)[³H]NECA [³H]ZM241385 Receptor (agonist) (antagonist) wt 32 ± 1 12 ± 3Rag 1   26 ± 0.4   26 ± 0.5 Rag 23 21 ± 1 62 ± 1 Rant 21 >450 15 ± 3Values are representative of three independent experiments. Each datapoint was assayed in triplicate and plotted as mean ± SD. Data werefitted to the Michaelis-Menten equation using the non-linear regressionequation of the software Prism.

TABLE (v) Summary of stability of wild-type and mutant receptors indifferent detergents. Tm (° C.) Agonist- Antagonist- binding binding wtRag 23 wt Rant 21  0.01% DDM 27 34 25 39  0.1% DM 23 29 10 28  0.3% NM22 28 <4 25  0.3% NG † † † 22  0.6% OG <9 16 † 23 0.003% LDAO 28 38 3242 0.006% FC12 37 39 43 49 Solubilisation of receptors and detergentexchange was performed during the IMAC step. S.D. is <1° C. It was notpossible to determine the Tm for some receptor-detergent combinations,because the receptor was too unstable (†).

EXAMPLE 3 Mutants of the Neurotensin Receptor (NTR) with IncreasedThermostability

-   1. 340 site-directed mutants have been made between residues 61-400    of NTR.-   2. Initially, all of these mutants were assayed for thermostability    using an assay measuring ³H-neurotensin (agonist) binding after the    heating step. 24 mutations led to a small but significant increase    in thermostability: A356L, H103A, D345A, A86L, A385L, Y349A, C386A,    K397A, H393A, I116A, F358A, S108A, M181A, R392A, D113A, G209A,    L205A, L72A, A120L, P399A, Y351A, V268A, T207A, A155L, S362A, F189A,    N262A, L109A, W391A, T179A, S182A, M293A, L256A, F147A, D139A,    S100A, K176A, L111A, A90L, N270A.-   3. Mutants tested for thermostability by heating in the absence of    the agonist were re-tested using a slightly different assay where    the mutants were heated in the presence of ³H-neurotensin (Ligand(+)    format in FIG. 12). Mutants with improved thermostability are: A69L,    A73L, A86L, A90L, H103A, V165A, E166A, G215A, V229A, M250A, 1253A,    A177L, R183A, 1260A, T279A, T294A, 0306A, L308A, V309A, L310A,    V313A, F342A, F358A, V360A, S362A, N370A, S373A, F380A, A385L,    P389A, G390A, R395A.-   4. There are also mutants that have a significantly enhanced    expression level compared to the wildtype receptor and could be used    to boost preceptor production levels for crystallisation: A86L,    H103A, F358A, S362A, N370A, A385L, G390A. All of these also have    increased thermostability.-   5. Preferred combinations are

a. Nag7m F358A + A86L + I260A + F342A Tm 51° C. (neurotensin bound) b.Nag7n F358A + H103A + I260A + Tm 51° C. (neurotensin F342A bound)

-   -   Wildtype NTR has a Tm of 35° C. with neurotensin bound.

EXAMPLE 4 Identification of Structural Motifs in which Stabilising GPCRMutations Reside

The structure of the β2 adrenergic receptor has been determined (20,21), which is 59% identical to the turkey β₁ receptor, but with adistinctly different pharmacological profile (22, 23). In order todetermine the structural motifs in which the stabilising mutations ofthe turkey β₁ receptor reside, we mapped the mutations onto the human β2structure (21).

The beta adrenergic receptors were first aligned using ClustalW in theMacVector package; thermostabilising mutations in turkey β1 werehighlighted along with the corresponding residue in the human β2sequence. The human β2 model (pdb accession code 2RH1) was visualised inPymol and the desired amino acids were shown as space filling models bystandard procedures known in the art. The structural motifs in which thestabilising mutations were located, were determined by visualinspection.

Table (vi) lists the equivalent positions in the β2 receptorcorresponding to the thermostabilising mutations in βAR-m23 and thestructural motifs in which they reside.

As seen from Table (vi), the mutations are positioned in a number ofdistinct localities. Three mutations are in loop regions that arepredicted to be accessible to aqueous solvent (loop). Eight mutationsare in the transmembrane α-helices and point into the lipid bilayer(lipid); three of these mutations are near the end of the helices andmay be considered to be at the hydrophilic boundary layer (lipidboundary). Eight mutations are found at the interfaces betweentransmembrane α-helices (helix-helix interface), three of which areeither within a kinked or distorted region of the helix (kink) andanother two mutations occur in one helix but are adjacent to one or moreother helices which contain a kink adjacent in space to the mutatedresidue (opposite kink). These latter mutations could affect the packingof the amino acids within the kinked region, which could result inthermostabilisation. Another mutation is in a substrate binding pocket(pocket).

TABLE (vi) Position in the human β2 structure of the amino acid residuesequivalent to the thermostabilising mutations found in the turkey β1receptor and the structural motifs in which they reside. Turkey β1 Humanβ2 Description Helix 1 I55A I47 3-helix kink interface FIG. 18 Helix 1G67A A59 lipid boundary Helix 1 R68S K60 lipid boundary FIG. 25 Helix 2V89L V81 kink FIG. 19 Helix 2 M90V M82 kink FIG. 20 Helix 2 G98A G90pocket Helix 3 I129V I121 opposite kink FIG. 21 S151E S143 loop Helix 4V160A V152 lipid Q194A A186 loop Helix 5 L221V V213 lipid Helix 5 Y227AY219 helix-helix interface FIG. 23 Helix 5 R229Q R221 lipid Helix 5V230A V222 helix-helix interface Helix 5 A234L A226 helix-helixinterface Helix 6 A282L C265 loop FIG. 24 D322A K305 lipid boundaryHelix 7 F327A L310 lipid Helix 7 A334L V317 lipid Helix 7 F338M F321kink FIG. 22

Such structural motifs, by virtue of them containing stabilisingmutations, are important in determining protein stability. Therefore,targeting mutations to these motifs will facilitate the generation ofstabilised mutant GPCRs. Indeed, there were several instances where morethan one mutation mapped to the same structural motif. For example, theY227A, V230A and A234L mutations in the turkey β1 adrenergic receptorall mapped to the same helical interface, the V89L and M90V mutationsmapped to the same helical kink and the F327A and A334L mutations mappedto the same helical surface pointing towards the lipid bilayer (Table(vi)). Thus, when one stabilising mutation has been identified, thedetermination of the structural motif in which that mutation is locatedwill enable the identification of further stabilising mutations.

EXAMPLE 5 Generation of Conformation Specific Binding Partners of GPCRs

The generation of mutant GPCRs having increased stability in aparticular conformation relative to a parent GPCR provides a number ofadvantages for screening of binding partners. For example, the presentmethods reduce the amount of material required for a screen. In standardscreens, GPCRs are present in whole cells or in membranes from wholecells which are usually screened by incubation with individual compoundsin isolated chambers rather than libraries of compounds. Therefore thepresent invention provides advantages in terms of time required to carryout a compound screen. The ability to lock a GPCR in a particularconformation provides advantages in that it increases the likelihood ofidentifying a ligand with the required pharmacological properties. Instandard binding screens GPCRs are able to assume a number of differentconformations and binding compounds will be identified across differentpharmacological types. Reagent costs can be reduced due to the abilityto miniaturize the assay formats and this is facilitated by the presentmethod.

Methods Compound Screening

A mutant GPCR having increased stability in a particular conformation isimmobilised on a solid surface and incubated with buffer containing anencoded library of compounds. After a suitable period of time, to allowbinding between the mutant GPCR and compounds from the library whichselectively bind to the mutant GPCR, the buffer is removed. Next therefollows a number of wash steps to remove compounds which have not boundspecifically to the mutant GPCR. The reading code, tag or address (suchas DNA or RNA) is then used to identify the small molecule bound eitherwhilst still bound to the mutant GPCR or following elution from theGPCR. The conformation specific binding partner is subsequentlyisolated.

Selection of Antibodies Using Immobilised GPCRS

Immobilised stabilised GPCRs can be used to select antibodies to thereceptor from mixtures of antibodies such as would be present in plasmafrom an animal immunised with the stabilised GPCR, the native receptoror a peptide from the receptor. Antibodies could be identified fromsupernatants obtained from B-cells taken from immunised animals or fromhybridomas obtained following immortalization of B-cells from theimmunised animal or from recombinant antibody libraries which may beexpressed on phage particles or through an in vitro expression systemsuch as ribosome display. The method has the advantage of selectingantibodies to particular conformations of a receptor. A stabilised GPCRlocked in the antagonist or ground state of the receptor would increasethe probability of selecting an antagonistic antibody whereas astabilised GPCR locked in the activated or R* state would increase theprobability of selecting an activating antibody. In standard screensantibodies are often selected to GPCRs that bind to peptide epitopes ofthe receptor but do not have antagonist or agonist properties andtherefore are not useful as therapeutic agents.

EXAMPLE 6 Antibody Phage Selection on β-Adregenic Receptor Summary

We have used stabilised β-adrenergic receptor (β-AR) to generateantibodies using phage display. Positive phage clones showed specificityfor β-AR and sub-cloning of selected antibody genes led to successfulproduction of anti-β-AR specific scFv antibodies.

Introduction

The therapeutic application of antibodies in the area of receptor-ligandsystems has great potential. However, the primary issue in generatingantibodies (either by in vitro or in vivo methods) to G protein coupledreceptors (GPCRs) concerns the immunogenic recognition of a specificconformation in a homogeneous antigen preparation, i.e. either anagonistic conformation or antagonistic conformation, rather thancreating a pool of antibody binders that recognises purified antigen inmultiple conformations in a heterogeneous preparation. In addition,recombinant receptor antigen is usually available only in theextracellular domain form, which precludes any tertiary structureinvolving other parts of the receptor. The proposed solution to thisissue is the application of stabilized GPCRs as the target antigen.

We have demonstrated the utility of stabilised GPCRs (StaRs™) in thegeneration of recombinant antibodies by the in vitro method of phagedisplay such that antibodies which bind the β adrenergic receptorstabilised in the antagonist conformation may be isolated. Suchantibodies can then be subject to functional assays e.g. in ligandbinding assays.

Overview of Process

A typical phage display strategy comprises several stages in the processto identify inhibitory scFv clones (FIG. 29). The first part is theselection of phage libraries on antigen to isolate a population of phageantibody binders using various methodologies (for example, panningselection, soluble selection, etc.). This resulting population of phageantibody binders is referred to as a selection output. This process isrepeated 2-4 times to enrich for specific antigen binders.

A pool of clones representing a selection output (polyclonal phageELISA) is assessed for recognition of antigen by phage ELISA. Individualclones can also be assessed by monoclonal phage ELISA and for diversityby sequence analysis, however the preferred method is to subclone theselected population into a recombinant antibody expression vector (pSANG10-3F) and then perform the assessment by monoclonal scFv ELISA and DNAsequencing. This circumvents the problem of identifying phage antibodybinders that subsequently exhibit poor expression as scFv fragments.Selection outputs yielding diverse ELISA positives can then beprioritised for functional screening of larger panels of phageantibodies.

Selection outputs are subjected to a screening campaign, employing afunctional high-throughput assay, of periplasmic extracts in order toidentify a population of scFv inhibitors. Lastly, the hits from thescreening campaign are profiled as scFv by further functional assays, aswell as IC50 analysis which assess the efficiency of their inhibitoryaction.

Methods and Results

2-3 rounds of antibody selections were carried out using stabilised β-ARas antigen and using the antibody phage display library described inSchofield et al, 2007 (24). Selections were carried out in PBS in thepresence (A) or absence (B) of 20 nM ligand (−)-alprenolol duringbinding and washing steps. Further, for β-AR protein handling, allcoating, washing and blocking buffers were supplemented with 0.1%detergent decylmaltoside (Anatrace, Anagrade). The relative success ofthe selectives was determined using polyclonal phage ELISA andmonoclonal phage ELISA.

Optimisation to Immobilise Antigen

Immobilisation strategies were based on taking advantage of the Histagged antigen. In the first instance, immobilisation was done usingcontrol proteins rather than βAR-m23. A variety of surfaces, includingNi-NTA plates, were employed in the initial assessment. All werecompared against standard passive absorption onto Nunc plates. In allcases, the outcome was evaluated using polyclonal phage ELISA after 2rounds of selection.

Antibody Selection

Selection, elution and rescue of the library was as described inSchofield et al (2007). 150 μl of β-AR at concentration of 20 μg/ml wascoated over night at +4° C. in two (24) wells of a Nickel chelate-plate(Nunc). The receptor was diluted in coating buffer which is 20 mM TrispH8, 100 mM NaCl, 0.1% decylmaltoside (dec-M) and also 20 nM ligand forselection A. Next day, the wells were rinsed with PBS and blocked for 1h at room temperature with 3% Marvel milk protein in PBS supplementedwith 0.1% dec-M (PBS-M). After coating, the well was rinsed in PBS and100 μl of phage library pre-blocked in 2% Marvel/PBS was added andincubated for 1 h at room temperature. Following binding, samples werewashed 6 times in PBS/0.1% Tween supplemented with 0.1% dec-M and sixtimes in PBS supplemented with 0.1% dec-M. Bound phage was then elutedwith Trypsin (24).

Eluted phage were added to exponentially growing TG1 cells (atOD₆₀₀=0.5) and grown at 37° C. for 1 h. Infected cells were plated ontoTY plates supplemented with 100 μg/ml ampicillin, 2% glucose and grownovernight at 30° C. Next day, plates were scraped in TY mediumsupplemented with 100 μg/ml ampicillin, 15% glycerol for storage. Thepopulation from this first round of selection was rescued with helperphage and PEG precipitated and 100 μl of these were used in a secondround of selections using the same conditions and procedures asdescribed for round 1.

Polyclonal Phage ELISA

For ELISA, βAR was covalently immobilised on Amino plates (Nunc CatalogNo: 436008). Coating and washing buffers were supplemented with 20 nMligand and 0.1% dec-M. The ELISA plate was coated overnight with β-AR at24 μg/ml and 2 control proteins (CD86 and Notch1) at 5 μg/ml. Next daythe wells were washed and blocked with PBS-M. 50 μl/well of polyclonalphage from 2 rounds of selection were added (in PBS-M, at aconcentration of 0.1× relative to the initial culture volume) andincubated for 1 hour at room temperature. Wells were washed andincubated with an α-M13 antibody (GE healthcare product No: 27-9421-01),for 1 hour at room temperature. Plates were washed and incubated withEuropium labelled anti-mouse antibody (Perkin Elmer, product No: AD0207), for 1 hour at room temperature. Wells were washed and 50 μl/wellof enhancement solution (Perkin Elmer, product No: 4001-0010) added andincubated for 10 min. The results are shown in FIG. 30A, which suggestsspecific enrichment of β-AR binding phage in experiments A and Bfollowing 2 rounds of selection. Signals were higher from phageselection A (with ligand present in buffers).

Monoclonal Phage ELISA

For this assay, individual clones of round 2 phage from selection A werepicked, rescued and PEG precipitated. Again, coating, washing, blockingand antibody detection buffers were supplemented with 20 nM ligand and0.1% detergent. Wells of a Nunc Amino plate were coated with β-AR for 1h 30 minutes at room temperature. Coated wells were washed 3 times withPBST and 3 times with PBS and blocked with PBS-M and 50 ul/well of 0.1×phage was added and incubated for 1 h at room temperature. Wells werewashed 6 times with PBS and incubated with α-M13 antibody for 1 h atroom temperature. After washing 6 times with PBS the wells wereincubated with anti-mouse-Eu antibody, 1 h at room temperature. Next,wells were washed and incubated with enhancement solution for 10 min.Out of 69 screened clones, 20 clones were detected as positive (FIG.30B).

Specificity Phage ELISA

17 of the positive phage clones from the monoclonal ELISA were testedfor binding specificity to β-AR and 3 unrelated proteins. This includesN1-EGF, (EGF domains 1-12 of murine Notch1 fused to a human Fc domainfrom R&D Systems, catalogue number 1057-TK). The other control proteinsinclude the extracellular domain of murine CD86 and the negativeregulatory region of murine Notch1 expressed as a CD4 fusion (CD86 andN1(NRR) respectively) in a transient expression system as described inChapple et al 2006 (25). In this ELISA both nickel chelate andamino-plate were used for comparison. Again, for wells containing β-ARprotein, coating, washing, blocking and antibody binding buffers weresupplemented with 20 nM ligand and 0.1% detergent. Wells were washed andincubated with antibodies as described for the monoclonal ELISA inprevious section. The assay showed that both His and Amino-plates can beused and that the majority of the anti-β-AR clones do not cross reactwith the unrelated proteins (FIG. 31).

Expression and Screening of Monoclonal scFv

Sub-cloning, antibody single chain Fv (scFv) expression and purificationwere as described in Schofield et al (2007). Selected antibody geneswithin the round 2 phage population (selection A) were sub-cloned intothe pSANG10-3F vector (26) and transformed into BL21(DE3) cells. 96colonies were picked and periplasmic expression of the scFv antibody wasinduced in a 96 well format using standard methods in the art. scFv wererecovered from the periplasm and used for ELISA on an Amino plate(Nunc). Washed and blocked wells were incubated for 1 h with 50 μl/wellof scFv. Plates were washed and incubated with Europium labelledanti-FLAG antibody for 1 h at room temperature. 12 clones gave a signalabove 1000 units with background levels of less than 50 for negativeclones (FIG. 32).

Positive clones are selected with binding and specifically confirmed ina secondary ELISA, and clones of interest sequenced. Antibody sequencesare analysed to identify the number of unique binding clones that havebeen isolated.

To test for blocking of ligand binding by isolated antibodies, uniquepositive antibody clones are selected for larger scale preparation(50-500 ml) using periplasmic extraction and immobilised metal affinitychromatography. This material is assessed for the ability to interferein ligand binding to receptor using robust reporter assays (e.g.inhibition of cAMP generation or inhibition of ligand binding totransfected cells).

EXAMPLE 7 Assessment of Compound Interaction with β1-AR Methods andResults:

Binding studies were performed at 10° C. using a Biacore S51 opticalbiosensor equipped with a streptavidin-coated CM5 chip and equilibratedwith running buffer (20 mM trisHCl, 150 mM NaCl, 1 mM EDTA, 1% DMSO,0.1% decyl maltoside, pH 7.8).

β1AR Immobilization.

β1AR36-M23 was minimally biotinylated using EZ-link sulfo-NHS-LC-LCbiotin (Pierce #21338): The biotin was added to the receptorpreparation, (spiked with 100 uM alprenolol) and allowed to react forthree hours at 4° C., after which free biotin was removed via columnchromatography.

FIG. 33 shows the two capture stages of the biotinylated b1AR onto astreptavidin-coated flow cell. In the first stage, we captured ˜1200 RU;in the second, 4000 RU.

The biacore sensor chips coated in the stabilised beta receptor could beused to characterise the binding of drugs with activity at the betareceptor. Flow through of the compound allowed the on rate to bedetermined. Subsequent washing enabled determination of the off rate.These parameters could then be used to determine a kinetic affinitymeasurement (Kd).

FIG. 34 shows the responses for alprenolol which was tested in replicatein a three-fold dilution series for binding to the receptor surface Thehighest concentration is 666 nM and each concentration was tested threetimes. The responses are concentration dependent and are reproducible.The grey lines depict the fit of a simple 1:1 interaction model and theparameters determined from this fit are listed in the inset (the numberin parentheses is the error in the last digit).

FIG. 35 is an alternative depiction of the data set in FIG. 34. Theresponses are normalized with respect to the Rmax determined (from thefitting) for each curve.

The alprenolol data demonstrates that the biotinylated b1AR is activeand this biosensor approach can be used to characterize thecompound/receptor interactions.

FIG. 36 shows the responses for propranolol binding to β1AR. 111 nM isthe highest concentration and each concentration was tested two or threetimes. The propranolol data are plotted as normalized responses in FIG.37.

When the β1AR surface was almost three days old, we retested alprenololbinding to determine how much activity the receptor had lost over time.FIG. 38 shows the alprenolol binding responses with 333 nM as thehighest concentration. The receptor appeared to be nearly as active aswhen it was first captured.

β1AR Capture on Another Spot.

The S51 biosensor has the ability to monitor two reaction spots at onetime so we captured the receptor to a density of 8000 RU on anotherstreptavidin-coated spot (FIG. 39).

The data from a test run of alprenolol binding to the 8000-RU b1AR spotare provided in FIG. 40.

The beta receptor agonist salmeterol was tested using a highestconcentration of 1.67 μM, with each concentration tested two or threetimes (FIGS. 41A and B). The responses are concentration dependent andmostly reproducible. Also, the responses from the 8000 RU surface (FIG.41B) were larger than from the 4200 RU surface (FIG. 41A), as expected.

Salmeterol dissociated much faster and bound the receptor much moreweakly than alprenolol or propranolol did. This is to be expected sincesalmeterol has a low affinity for the β1-AR and in particular has a lowaffinity for the antagonist stabilised form of the receptor which isused here. In addition, we detected some complexity in the interaction,as indicated by the poor fit of the model to the responses during thedissociation phase (t>60 sec). FIGS. 42A and B shows the data plotted asnormalized responses.

Isoproterenol, a non-selective β-adrenergic agonist, was tested using ahighest concentration of 2 μM (FIGS. 43A and B). In this analysis,isoproterenol displayed a much slower association rate than the othercompounds. The compound was injected for 90 sec.

FIGS. 44A and B show responses for 111 uM and 333 nM carvedilol bindingto the two b1AR surfaces. Carvedilol displays a much slower dissociationrate than the other compounds examined so far.

Conclusion

-   -   1. Minimal biotinylation and capture by streptavidin produced        active b1AR surfaces that can be used to measure compound        binding.    -   2. At 10° C., the b1AR surfaces remained active over several        days.    -   3. For the compounds tested including agonists and antagonists,        we observed differences in both the association and dissociation        rates, as well as the affinities. This demonstrates the        biosensor assay is a viable approach to characterising panels of        compounds binding to this b1AR preparation.    -   4. Binding parameters were determined for several compounds as        shown in Table A below, all of which were measured at 10° C.

TABLE A k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (nM) Alprenolol (1.453 ± 0.007)× 10⁵ (6.3 ± 0.1) × 10⁻³ 44 ± 1  Propranolol  (5.94 ± 0.02) × 10⁵ (2.17± 0.04) × 10⁻³  3.64 ± 0.07 Salmeterol¹   (3.8 ± 0.3) × 10⁴ (9.5 ± 0.4)× 10⁻² 2500 ± 300  Isoproterenol^(1,2)      (6 ± 2) × 10³ (4.4 ± 0.2) ×10⁻³ 800 ± 300 Carvedilol^(1,2)   (2.3 ± 0.9) × 10⁵ (1.1 ± 0.1) × 10⁻³ 5± 3 ¹averaged from two b1AR surfaces ²preliminary results

EXAMPLE 8 Use of the Adenosine A2a StaR for Compound Screening in DrugDiscovery Methods

A thermostabilised adenosine A2a receptor (A2a StaR) conformationallyselected in the antagonist form (referred to as Rant22) was used toscreen compounds from a library in order to identify compounds withactivity at the A2a receptor. The StaR was generated as previouslydescribed (Magnani et al, Co-evolving stability and conformationalhomogeneity of the human adenosine A2a receptor. Proc Natl Acad Sci USA.2008 Aug. 5; 105 (31):10744-9). HEK293T cells transfected with Rant22A2a receptors were grown in a monolayer in T-175 flasks at 37° C. and 5%CO₂ in Dulbeccos Modified Eagle Medium supplemented with 10% fetalbovine serum. Cells were harvested by scraping the cells from the T-175surface and collected by centrifugation.

Membrane Preparation.

Cell pellets were resuspended in 10 ml 20 mM HEPES, pH 7.4 plus proteaseinhibitor cocktail tablets (Roche) and were homogenised for 30 s at20,500 rpm using a tissuemizer. Homogenates were centrifuged at 200×gfor 15 min at 4° C.

The supernatant was removed and reserved on ice. This procedure wasrepeated a further two times and the pooled supernatants were thencentrifuged at 40,000×g for 45 min at 4° C. Membranes were resuspendedin 1 ml aliquots of 20 mM HEPES, pH 7.4 plus protease inhibitor tablets.Protein concentration was determined by a BCA protein assay (Pierce).

Screening Assay.

10 μg aliquots of prepared membrane were incubated with [³H]ZM241385 3.7nM and an appropriate amount of unlabelled ligand for 60 min at roomtemperature. A serial dilution ranging from 10 mM-1 μM was screened in a96 well format. The filter GFC plates were pre soaked in 0.1% PEI for 60min. Radioactivity was determined by liquid scintillation counting usinga Microbeta counter at 3 min/well.

Results

Data was analysed using GraphPad prism to fit concentration responsecurves. The IC₅₀ of compounds was calculated as the concentrationresulting in 50% inhibition of the specific binding of [³H]ZM241385. Thedata presented in FIG. 45 and in Table B below demonstrates that thecompounds tested were able to inhibit binding to the A2a receptor StaRand had a range of activities in this assay. This data demonstrates theutility of StaRs for compound screening.

TABLE B HTL0245 HTL0246 HTL0247 HTL0248 HTL0249 Best-fit values Bottom105.2 151.2 47.23 44.21 25.74 Top 1428 1414 1406 1519 1527 LogIC50−6.795 −6.442 −7.552 −8.098 −8.431 IC50 1.605e−007 3.614e−007 2.804e−0087.985e−009 3.709e−009 Std. Error Bottom 29.07 70.00 40.67 18.05 7.115Top 44.68 83.73 96.91 50.84 20.46 LogIC50 0.07888 0.1886 0.1338 0.052050.02427 HTL0250 HTL0251 HTL0252 CGS15943 Best-fit values Bottom 13.98−5.831 176.4 22.40 Top 1382 1446 1257 1530 LogIC50 −4.112 −2.874 −4.658−8.661 IC50 7.735e−005 0.001337 2.199e−005 2.183e−009 Std. Error Bottom104.4 79.15 154.9 29.97 Top 38.13 26.10 74.89 40.93 LogIC50 0.12020.2821 0.2878 0.08677

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1. A method of producing a binding partner of a GPCR, the methodcomprising: a) providing a mutant GPCR of a parent GPCR, wherein themutant GPCR has increased stability in a particular conformationrelative to the parent GPCR; b) providing one or more test compounds; c)determining whether the or each test compound binds to the mutant GPCRwhen residing in a particular conformation; and d) isolating one or moretest compounds that bind to the mutant GPCR when residing in theparticular conformation.
 2. A method according to claim 2, the methodfurther comprising: e) determining whether the or each test compoundbinds to the parent GPCR when residing in the particular conformationand f) isolating the or each test compound that also binds the parentGPCR when residing in the particular conformation.
 3. A method accordingto claim 1, wherein the mutant GPCR is immobilised onto a solid support.4.-33. (canceled)
 34. A method according to claim 1, wherein more thanone mutant GPCR is provided in step (a), it is determined whether thetest compound binds to each mutant GPCR when residing in a particularconformation; and the test compound which binds to each mutant GPCR whenresiding in the particular conformation is isolated.
 35. (canceled) 36.A method according to claim 1, wherein more than one mutant GPCR isprovided in step (a) and a test compound is selected that binds to afirst mutant GPCR but which does not bind, or binds less strongly thanto the first mutant GPCR, to at least one other mutant GPCR.
 37. Amethod according to claim 1, further comprising (i) determining if theisolated test compound affects the function of the GPCR to which itbinds and (ii) isolating a test compound that affects the function ofthe GPCR to which it binds.
 38. A method according to claim 37, whereinin step (i) or step (ii) it is determined if the isolated test compoundaffects the binding of the GPCR to its natural ligand or analog thereof.39. (canceled)
 40. A method according to claim 38, wherein in step (ii)a test compound that increases or decreases binding between the GPCR andits natural ligand or analog thereof is isolated.
 41. A method accordingto claim 37, wherein in step (i) it is determined if the isolated testcompound modulates activation of the GPCR to which it binds.
 42. Amethod according to claim 41, wherein in step (ii) a test compound whichmodulates any of calcium mobilisation, cAMP levels, kinase pathwayactivity, gene transcription from a reporter gene under control of theGPCR to which the test compound binds, β-arrestin recruitment,activation of G proteins, GTPase activity or [35S]GTPγS binding isselected.
 43. A method according to claim 41, wherein in step (ii) anagonist test compound that increases activation of the GPCR to which itbinds, is isolated.
 44. A method according to claim 41, wherein in step(ii) an antagonist test compound that decreases activation of the GPCRto which it binds, is isolated.
 45. A method according to claim 1,wherein the mutant GPCR is provided by: (a) providing one or moremutants of a parent GPCR, (b) selecting a ligand, the ligand being onewhich binds to the parent GPCR when the GPCR is residing in a particularconformation, (c) determining whether the or each mutant GPCR hasincreased stability with respect to binding the selected ligand comparedto the stability of the parent GPCR with respect to binding that ligand,and (d) selecting those mutants that have an increased stabilitycompared to the parent GPCR with respect to binding the selected ligand.46.-60. (canceled)
 61. A method according to claim 1, wherein the mutantGPCR is provided by: (a) providing one or more mutants of a parent GPCR,(b) selecting a ligand, the ligand being one which binds to the parentGPCR when the GPCR is residing in a particular conformation. (c)determining whether the or each mutant GPCR has increased stability withrespect to binding the selected ligand compared to the stability of theparent GPCR with respect to binding that ligand, (d) selecting thosemutants that have an increased stability compared to the parent GPCRwith respect to binding the selected ligand, (e) identifying theposition or positions of the mutated amino acid residue or residues inthe mutant GPCR or GPCRs which has been selected for increasedstability, and (f) synthesising a mutant GPCR which contains areplacement amino acid at one or more of the positions identified. 62.(canceled)
 63. A method according to claim 1, wherein the mutant GPCR isprovided by: (i) identifying in the amino acid sequence of one or moremutants of a first parent GPCR with increased stability relative to thefirst parent GPCR, the position or positions at which the one or moremutants have at least one different amino acid residue compared to thefirst parent GPCR, and (ii) making one or more mutations in the aminoacid sequence that defines a second GPCR at the corresponding positionor positions, to provide one or more mutants of a second parent GPCRwith increased stability relative to the second parent GPCR. 64.-66.(canceled)
 67. A method according to claim 1, wherein of producing amutant GPCR with increased stability relative to its parent GPCR, themethod comprising: (i) providing one or more mutants of a first parentGPCR with increased stability relative to the first parent GPCR (ii)identifying in a structural membrane protein model the structural motifor motifs in which the one or more mutants have at least one differentamino acid residue compared to the first parent GPCR, and (iii) makingone or more mutations in the amino acid sequence that defines acorresponding structural motif or motifs in a second parent GPCR, toprovide one or more mutants of a second parent GPCR with increasedstability relative to the second parent GPCR. 68.-77. (canceled)
 78. Amethod according to claim 63, further comprising: (I) selecting aligand, the ligand being one which binds to the second parent GPCR whenthe GPCR is residing in a particular conformation (II) determiningwhether the or each mutant of the second parent GPCR when residing in aparticular conformation has increased stability with respect to bindingthe selected ligand compared to the stability of the second parent GPCRwhen residing in the same particular conformation with respect tobinding that ligand, and (III) selecting those mutants that have anincreased stability compared to the second parent GPCR with respect tobinding the selected ligand. 79.-96. (canceled)
 97. A method ofproducing a binding partner of a GPCR, the method comprisingsynthesising a binding partner identifiable by carrying out the methodof claim
 1. 98.-102. (canceled)
 103. A biosensor comprising a mutantGPCR of a parent GPCR, wherein the mutant GPCR has increased stabilityin a particular conformation relative to the parent GPCR and whereinwhen a target substance binds to said mutant GPCR, a detectable signalis produced.
 104. A biosensor according to claim 103, wherein the mutantGPCR is provided by: (a) providing one or more mutants of a parent GPCR,(b) selecting a ligand, the ligand being one which binds to the parentGPCR when the GPCR is residing in a particular conformation, (c)determining whether the or each mutant GPCR has increased stability withrespect to binding the selected ligand compared to the stability of theparent GPCR with respect to binding that ligand, and (d) selecting amutant that has an increased stability compared to the parent GPCR withrespect to binding the selected ligand. 105.-108. (canceled)